Optokinetic nystagmus (OKN) assists stabilization of the retinal image during head rotation. OKN is driven by ON direction selective retinal ganglion cells (ON DSGCs), which encode both the direction and speed of global retinal slip. The synaptic circuits responsible for the direction selectivity of ON DSGCs are well understood, but those sculpting their slow-speed preference remain enigmatic. Here, we probe this mechanism in mouse retina through patch clamp recordings, functional imaging, genetic manipulation, and electron microscopic reconstructions. We confirm earlier evidence that feedforward glycinergic inhibition is the main suppressor of ON DSGC responses to fast motion, and reveal the source for this inhibition—the VGluT3 amacrine cell, a dual neurotransmitter, excitatory/inhibitory interneuron. Together, our results identify a role for VGluT3 cells in limiting the speed range of OKN. More broadly, they suggest VGluT3 cells shape the response of many retinal cell types to fast motion, suppressing it in some while enhancing it in others. The optokinetic reflex assists image-stabilization in visual systems. Here the authors show that the slow speed preference of ON direction-selective ganglion cells, triggering optokinetic nystagmus, relies on inhibition from VGluT3 amacrine cells. Both produce nystagmus, featuring slow image-stabilizing eye movements periodically interrupted by fast resets of gaze in the opposite direction.Image-stabilizing reflexes supporting vision are extremely widespread among animals. In vertebrates, rotation of the head triggers vestibular and visual feedback signals to brainstem and cerebellum oculomotor networks. These generate eye counter-rotation to cancel the global drift or ‘retinal slip’ of the image. These innate responses comprise the vestibulo-ocular reflex (VOR) and the optokinetic reflex (OKR)Vestibular and visual image-stabilizing reflexes complement one another in the velocity domain. The VOR nulls most of the retinal slip during rapid rotatory head motion; the modest and slow residual slip triggers the OKR, improving the stabilization. During slow head rotation, when VOR gain is weak, OKR is the dominant stabilization mechanism. These two complementary reflexes sum to almost perfectly stabilize the retinal image over the physiological range of head rotations. The OKR is speed-tuned. It must be responsive to slow slip velocities to compensate for the limitations of the VOR, but must be insensitive to fast speeds lest it interfere with fast eye movements, such as saccades or the fast resetting phase of nystagmus.6, only a very specific subset is dedicated to visually driven reflexive image stabilization. These comprise a specific subclass of direction-selective ganglion cells (DSGCs), the ON DSGCs. They send their axons almost exclusively to the nuclei of the accessory optic system, which relays their retinal slip signals to the vestibulocerebellum and brainstem oculomotor centers7. This circuit is highly conserved across vertebrates, including apparently homologous ON DSGCs8. The far more common ON-OFF class of DSGCs makes only a small contribution to this circuit, projecting instead mainly to the lateral geniculate nucleus and superior colliculus.Among the ~40 types of mammalian retinal ganglion cells (RGCs)10. They are effectively silent during fast visual motion, as occurs during saccades or the fast phase of nystagmus. By contrast, ON-OFF DSGCs respond well to such fast motion10 (but see ref. 11). Other types of RGCs exhibit diverse speed tuning profiles, with some highly responsive to very fast motion.ON DSGCs, like the OKR they drive, respond best to slow speeds of retinal slip, typically ~1°/s, depending on species and stimulus15. By contrast, much less is understood about the cell types, neurotransmitters, and synaptic circuits that confer slow speed tuning upon ON DSGCs or shape the speed preferences of other RGC types.ON DSGCs thus encode both components of the vector of retinal slip—speed and direction—as required for image stabilization. Starburst amacrine cells (SACs) confer direction selectivity on these and other DSGCs through well-studied synaptic mechanisms16 or intrinsic filtering of the high-frequency excitatory events induced by fast motion. Alternatively, fast motion might trigger inhibition. This could act presynaptically, by suppressing the cell’s glutamatergic inputs from bipolar cells and from VGluT3 amacrine cells17. Alternatively, fast motion could evoke direct feedforward inhibition onto the ON DSGC itself. Indeed, this is the key mechanism for slow-speed preference in ON DSGCs of rabbits, with glycine serving as the main inhibitory transmitter10. ON-OFF DSGCs, a closely related class of direction-selective neurons, lack this glycinergic input and respond well to fast speeds10.Diverse mechanisms could generate slow-speed tuning in ON DSGCs. Excitatory drive might falter at higher speeds due to suboptimal spatiotemporal summation of excitatory inputs across the dendritic arbor10. Our connectomics studies identify an unexpected source of this glycinergic inhibition. It is the VGluT3 amacrine cell, an unusual interneuron that makes excitatory (glutamatergic) synapses onto some postsynaptic targets and inhibitory (glycinergic) synapses onto others20. Our functional data confirm that VGluT3 cells are the source of glycinergic inhibition onto ON DSGCs during fast motion. They also show that glutamatergic inputs from VGluT3 cells to ON-OFF DSGCs  augment their responses to fast motion. The connectomic data document widespread VGluT3 output to most types of RGCs. These amacrine cells may therefore play a major role in sculpting retinal output during fast motion by enhancing the responses of some RGCs and inhibiting others.Here in mouse retina we have used serial block face electron microscopy (SBEM), patch recordings, dendritic calcium imaging, and cell-type-specific optogenetics and chemogenetics to delineate the synaptic networks responsible for vetoing ON DSGC responses at high speeds. Our observations in mice echo those in the rabbit: direct feedforward glycinergic inhibition of ON DSGCs is primarily responsible2 or Pcdh9-Cre21) or by surveying RGCs for characteristic extracellular spike responses to flashed stimuli , close to previously reported optima for ON-DSGC responses and OKN gain in mice2. This probably slightly overestimates the mean preferred speed: About a quarter of the recorded cells responded best to the slowest speed tested (76 µm/s) and may have preferred even slower speeds. Spike responses were strongly attenuated at speeds above the optimum, dropping to half their maximum spike rate at roughly twice the preferred speed .As expected, ON DSGCs were remarkable among RGCs in preferring slower speeds of grating drift Fig. , blue. Sn = 17), Fig. 22, shared the slow speed tuning of ON DSGCs.Many other RGC types responded to much higher speeds of grating drift Fig. . For exa10. To test this idea, we first used whole-cell voltage clamp to record excitatory and inhibitory currents evoked in ON DSGCs by various speeds of grating drift in the preferred direction , or 24 °/s; peak charge transfer 490 ± 70 pC). Excitation declined to half its maximum at 2290 ± 170 µm/s. Thus, excitatory drive is tuned for speed, but with a very different profile than for spiking, implicating other mechanisms in the speed tuning of ON-DSGC output.We hypothesized that the slow-speed preference of mouse ON-DSGCs results from direct glycinergic inhibitory input, as demonstrated in rabbit retinan = 12; peak charge transfer 2200 ± 230 pC), it was weaker than excitation at slow speeds and stronger than excitation at fast speeds. The two normalized curves crossed at ~700 µm/s. At speeds faster than this, nearly all spiking was suppressed , n = 3. In contrast, blockade of GABAA receptors alone (SR95531) had only a modest effect . Simultaneous application of both antagonists abolished all inhibitory current in the ON DSGCs. Thus, effectively all inhibition driven by fast motion is mediated by GABAA and glycine receptors under our experimental conditions.We repeated these experiments adding receptor antagonists to the bath to test the hypothesis that the feed-forward inhibition evoked by fast motion was glycine-mediated, as in rabbit Fig. . Strychn10 and confirms that glycinergic inhibition plays a key role in limiting responses to rapid motion . In the presence of strychnine, ON DSGC firing could track the high temporal frequencies associated with fast grating motion , an amacrine-cell type that releases both glutamate and glycine as neurotransmitters. Convergent anatomical evidence supporting the identity of the reconstructed neurons as VGluT3 cells is provided in Supplementary Note 31 (2 synapses) or to an unidentified medium-field type (2 synapses). The cellular source of a few remaining contacts (22 of 460 synapses) had too few dendrites within the volume to be identified. We conclude that VGluT3 amacrine cells are by far the best candidate source of glycinergic inhibition shaping the slow-speed tuning of ON DSGCs.To identify the sources of glycinergic suppression of ON-DSGCs during fast motion, we turned to serial electron microscopic reconstruction. In a published SBEM dataset of the adult mouse inner plexiform layer spanning >200 μm17. To test for the inferred glycinergic influence, we recorded postsynaptic currents evoked in ON DSGCs by selective optogenetic depolarization of VGluT3 cells. For these studies, we crossed a strain of VGluT3-Cre mice with a strain (Ai32) that expresses channelrhodopsin2 (ChR2) in a Cre-dependent manner .The connectomic analysis revealed that a single type of glycinergic amacrine cell—the VGluT3 cell—supplies virtually all of the glycinergic synaptic contacts onto ON DSGCs. The VGluT3 amacrine cell is thus the presumed source of the glycinergic suppression of ON DSGCs during rapid motion. This is a surprising conclusion because VGluT3 cells have been reported to provide glutamatergic excitation to ON DSGCs, not glycinergic inhibitionn = 13 cells; three cells were excluded due to atypical or inconsistent currents, see “Methods” and Supplementary Note Optogenetic activation of VGluT3 cells evoked robust inhibitory currents in practically all ON DSGCs tested. Peak inhibitory postsynaptic currents averaged 43 ± 7 pA and 26 ± 6 pA, for single light pulses with durations 1 s and 0.1 s, respectively  had little effect on the evoked inhibitory conductance  light pulses . All ON DSGCs exhibiting such evoked excitation also exhibited optogenetically evoked IPSCs when clamped at +20 mV (n = 8). Optogenetically evoked IPSC and EPSC measured in the same cells correlated positively . This is consistent with both currents arising from VGluT3 cells and variable success at driving that dual input optogenetically.To probe the previously reported glutamatergic excitation of ON DSGCs by VGluT3 cellsp = 0.012, n = 3) for viral experiments and 38 ± 6%  for those based on the genetic cross. The same ligand had no effect on inhibition of ON DSGCs in a DREADD-free mouse . Thus, the native inhibition in ON DSGCs at high speeds is provided at least to a large extent by glycine release from VGluT3 cells.To test the idea that VGluT3 cells are the source of the inhibition that suppresses ON DSGC during fast motion, we studied the effects of chemogenetic suppression of VGluT3 cells on this inhibition. We used a DREADD system (Designer Receptor Exclusively Activated By Designer Drugs) in VGluT3-Cre mice, expressing the hM4Di receptor in VGluT3 cells either by intraocular injection of a Cre-dependent viral vector or by crossing VGluT3-Cre mice with Cre-dependent DREADD mice (“Methods”). In either case, bath-application of the DREADD ligand (CNO) markedly reduced the inhibitory currents induced by drifting gratings at all speeds Fig. . At speeThe chemogenetic approach was less effective than strychnine in reducing the motion-induced inhibition Fig. . Additio32. We saw no sign of inhibition. Thus, VGluT3 cells apparently exert mainly opposing influences on the two DSGC classes. ON-OFF DSGCs responded well to gratings moving at speeds that induced strong glycinergic suppression in ON DSGCs VGluT3 dendrites exhibited robust calcium signals in response to the motion of full-field grating stimuli, especially at the fast speeds that trigger feedforward glycinergic inhibition of ON DSGCs Fig. , and at 34. Indeed, calcium responses were greatly enhanced when we restricted the same gratings to a circular patch centered on the FOV . Slow gratings that failed to drive responses when presented full-field now evoked clear Ca2+ signals. Nonetheless, the high-speed preference remained.The robust response to extended stimuli was unexpected because VGluT3 receptive fields have strong suppressive surrounds34 . Further, the optimum stimulus size for gratings was 50% larger than for spots . Grating stimuli differed from spots in contrast as well as in motion, and both of these contributed to the difference in the observed strength of surround suppression . On the other hand, many VGluT3 ROIs preferred motion in one direction; nearly two thirds of them (65%) met the standard criterion for direction selectivity . Only 6% (15 ROIs) showed neither orientation nor direction preference. Over half (54%) met both criteria. In some FOVs, ROIs were either mostly orientation selective . Preferred directions of DS ROIs clustered mainly near one of the two directions along the same axis as the main cluster of preferred orientations . Note that we defined the preferred orientation as the preferred axis of motion .To our surprise, VGluT3 dendritic Caion Fig. . When prion Fig. ; 84% of ive Fig.  or mostlive Fig. , left, wive Fig. , right. Surprisingly, the direction and orientation selectivity we observed in VGluT3 dendritic calcium responses was not clearly evident in the glycinergic inhibitory current in ON DSGCs under pharmacological manipulation (Supplementary Note 35). It features a familiar amacrine cell type—the VGluT3 cell. We show that VGluT3 cells respond well to fast global motion, and that they veto ON DSGC responses to such motion through feedforward glycinergic inhibition.We have identified the inhibitory synaptic circuit primarily responsible for speed selectivity in ON DSGCs in mice, and therefore in OKN  synaptic contacts onto ON DSGCs. Among these cell types, only the VGluT3 cell is glycinergic. We show that the VGluT3 plexus is strongly activated by rapid motion, and that this is true even during global retinal slip, despite the strong suppressive surrounds of VGluT3 receptive fields. We demonstrate that optogenetic activation of VGluT3 cells evokes glycinergic inhibitory currents in ON DSGCs whereas chemogenetic suppression of VGluT3 output reduces the inhibition of ON DSGCs triggered by rapid motion. The role of glycinergic inhibition in suppressing ON-DSGCs responses at high speeds has recently been confirmed in mouse17, not glycinergic inhibition37. We confirmed weak VGluT3-mediated excitation of ON DSGCs, but inhibition was much stronger. The discrepancy from the earlier work may be traceable our 20-fold dimmer optogenetic stimulus. As far as we are aware, this is the first example of any neuronal type receiving both excitatory and inhibitory input from VGluT3 cells. This is reminiscent of the mixed excitatory and inhibitory inputs from SACs to DSGCs38. Our reconstructions reveal that SAC and VGluT3 inputs account for more than 92% of the total inhibitory synapses onto ON DSGCs. Both SACs and VGluT3 cells have been found in higher primate retinas41, so the same two cell types may encode the retinal slip vector in the human retina. Genetic defects perturbing this circuit disrupt normal image stabilization in human patients3.The output of ON DSGCs to the accessory optic system encodes both components of the local vector of global retinal slip: direction and speed. The directional coding derives mainly from a specific amacrine-cell type, the SAC, through spatially asymmetric feedforward GABAergic inhibition of the ON DSGC. Here, we argue that VGluT3 cells specify the other vector component—speed. These dual-transmitter interneurons excite some RGCs through glutamate release, while inhibiting others through glycine. Two earlier studies reported only excitatory glutamatergic transmission from VGluT3 cells to ON DSGCs9. The mechanistic basis of this difference remained obscure until work in rabbit showed that only the ON DSGCs receive fast-speed glycinergic inhibition10. Here we confirm this in mice and identify VGluT3 cells as the source of inhibition. We also find that VGluT3 cells further differentiate the speed preferences of the two DSGC classes by preferentially augmenting fast-motion responses in ON-OFF DSGCs though excitatory glutamatergic synapses.It has been known for decades that ON DSGCs prefer slow speeds while ON-OFF DSGCs prefer faster speeds42 to ON DSGCs. If H18 cells are glycinergic, as suggested by their relatively small, highly branched dendritic arbors, their contributions to ON DSGC inhibition would have been blocked by strychnine but not by our chemogenetic manipulation. However, the transmitter composition of H18 cells is unknown, and they synapse onto ON DSGCs only very rarely. We therefore favor an alternate explanation—that VGluT3 cells are virtually the sole glycinergic input, but that we were only partly successful in chemogenetically blocking that input. This could have occurred because weak DREADD expression or biophysical factors did not allow complete chemogenetic suppression of VGluT3 glycine release. Partial suppression of VGluT3 cells could account for the finding that the CNO-sensitive inhibition was a fixed fraction of the total inhibition across speeds, and that the shapes of the inhibitory current traces were preserved following CNO application . However, SACs do not respond to optogenetic activation of VGluT3 cellsThe connectomic evidence suggests that the direct influences of VGluT3 cells extend far more widely to other retinal neurons than appreciated in earlier functional surveys  is likely to extend broadly to the retinal output. For RGC types getting excitatory glutamatergic drive from VGluT3 cells, we would expect boosted sensitivity to relatively fast motion. OFF transient alpha cells are one such type and our data confirm their high-speed sensitivity. When the VGluT3 contribution is inhibitory, we expect it to favor responses to slow speeds in the postsynaptic ganglion cells, as shown here for ON DSGCs. Just such a functional influence seems to be detectable in a bistratified medium-field RGC type variously termed ON-delayedGCs Fig. .2+ imaging revealed robust responses to global motion in VGluT3 dendrites. This is unexpected because VGluT3 cells are known to be strongly suppressed by visual stimulation of their receptive-field surrounds34. Responsiveness to global motion also seems at odds with the reported selectivity of these cells for local object motion34. We have shown that the strength of surround suppression is context dependent, as is often the case48. When the receptive field is probed with a moving grating of various sizes, rather than with flashed spots17, the surround appears weaker and the center appears larger Hze/J, #028534) was crossed with each of four Cre-dependent mouse lines: Ai32 26Sortm32(CAG-COP4*H134R/EYFP)Hze/J, #024109) for optogenetics; DREADD mice 26Sortm1Ute/J, #026219) for chemogenetics, Ai14 26Sortm14(CAG-tdTomato)Hze/J, #007908) for fluorescent labeling for characterization of the VGluT3-Cre mouse, and Ai148 Hze/J, #030328) for Ca2+ imaging. For the Müller cell control experiment, GLAST-Cre mice 1Nat/J, #012586) were crossed with Ai32.All procedures were in accordance with the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee at Brown University. We studied adult mice of either sex, 2–8 months old. Mouse sex was not recorded. Mice were housed in a 12 light/12 dark cycle at room temperature (22 °C), 40–60% relative humidity. Wild-type mice were C57BL/6J (Jackson Laboratory). To target ON DSGCs for recording, we used HoxD10-GFP mice LT174Gsat/Mmucd, MMRRC #032065) and Pcdh9-Cre mice NP276Gsat/Mmucd, MMRRC #036084). The VGluT3-Cre line . Under dim red light, the globe was incised, and the cornea, lens and vitreous humor were removed. A relieving ventral cut was made in the eyecup, and the retina was isolated. Three more cuts were made in the retina, roughly along the temporal, nasal and dorso-nasal directions. These were made asymmetrically to allow for disambiguation of retinal orientation. The retina was flat-mounted on a polylysine coverslip , which was secured in a recording chamber.Isolation of the retina was performed similarly to ref. 50. The animals were used in experiments three weeks after the last tamoxifen injection.Tamoxifen (Sigma-Aldrich) was dissolved in corn oil , sonicated  and placed in hot water  and once homogenous, passed through a 0.2 µm filter. Tamoxifen was injected IP, 2–2.5 mg per mouse, once a day for 3 days. This resulted in dense YFP labeling of Müller glia in GLAST-Cre x Ai32 micePatch-clamp recordings of isolated flat-mount retina were performed under voltage-clamp using a Multiclamp 700B amplifier, Digidata 1550 digitizer, and pClamp 10.5 data acquisition software . Pipettes were pulled from thick-walled borosilicate tubing . Retinas were continuously superfused during experiments with oxygenated Ames’ medium at 32 °C, flow rate ~5 ml/min. For cell attached recordings, Ames filled pipettes were used (tip resistance of 4–5 MΩ). For whole-cell voltage clamp recordings, pipettes filled with cesium internal solution  were used (tip resistance of 5.5–6.5 MΩ). To isolate excitatory and inhibitory synaptic currents, the recorded cell was held near the reversal potential for inhibition (~ −65 mV) and excitation (~ + 20 mV), respectively. Application of synaptic blockers to the bath as well as Clozapine-N-Oxide (CNO) was done by switching the perfused medium into medium containing the blocker and waiting for ~7 minutes. The blockers used were: strychnine , SR95531 , L-AP4 , ACET , Hexamethonium , CNQX , D-AP5 . In the DREADD experiments, CNO was used to activate the DREADD .49. Patterned visual stimuli, synthesized by custom software using Psychophysics Toolbox under Matlab (The MathWorks), were projected  and focused onto the photoreceptor outer segments through the microscope’s condenser. The projected display covered ~1.5 × 1.5 mm (5.8 µm/pixel). The video projector was modified to use a single UV LED lamp . The LED’s spectrum (385 ± 5 nm) shifted to a 395 ± 12 nm peak skewed towards shorter waelengths after transmission through the projector and condenser optics, as well as a 440 nm band-pass filter , and various reflective neutral density filters (Edmund Optics), and reflection at a dichroic mirror . The photoisomerization rates used were 2–5 × 103 R*/rod/s (1–2 × 10−2 W·m−2), and for the spectrum of the stimulus were similar among rods, M-cones and S-cones (see ref. 46 and references therein). The ratios between photoizomerization rates for rods, M- and S-cones depend solely on the spectrum of the light source, and were 2.5: 2: 1.7, respectively. In the beginning of a stimulus sequence, a uniform screen with the stimulus’ mean intensity (gray) was projected for 20–30 s for light adaptation. To identify RGC types, the spike responses of the cell to a 460 µm diameter spot of +0.95 contrast were recorded. To assess the directional tuning of ON DSGCs we used  full-field sinusoidal gratings  drifting in 8 directions in a randomized sequence . For speed response curves, the same grating was drifted in the preferred direction (DSGCs), at 7–8 different speeds at a randomized sequence, with 3 repetitions for each speed. For VGluT3 dendrites Ca2+ imaging, the same stimulus was used, with the direction of motion chosen arbitrarily. The same was also presented in a circular patch on a gray background in Fig. Light stimuli were generated as in refs. p > 0.05, p < 0.05, p < 0.01 and p < 0.001, respectively.Throughout the text, we list group data as mean ± standard error of the mean, unless otherwise specified. All data analysis was done using custom written Matlab procedures. Individual peri-stimulus time histograms (PSTH) presented for spikes, or current traces for voltage clamp recordings, were averaged over three trials. For speed tuning curves ; Rabbit anti-VGluT3 . Chicken anti-GFP  was used to enhance the fluorescence of the Cre-dependent GFP virus. Rabbit anti-HA tag  was used to stain the HA-tagged hM4Di receptor in the VGluT3 x DREADD mouse. Secondary antibodies: Donkey anti-Chicken 488 ; Donkey anti-Chicken 594 ; Donkey anti-Goat 488 ; Donkey anti-Goat 594 ; Donkey anti-Rabbit 647 .https://imagej.net/software/fiji), and collapsed using either maximum intensity or maximum standard deviation projections.To target fluorescent cells for patch recording, two photon imaging was used (Olympus FV1200MPE BASIC (BX-61WI) microscope, 25×, 1.05 NA water-immersion objective , an ultrafast pulsed laser  tuned to 910 nm, and the imaging software Fluoview 4.1 (Olympus)). To acquire an image stack, RGCs were filled during electrophysiological recordings with Alexa hydrazide 488 or 594 , and were imaged following the recording, either using the two-photon or the single-photon  configuration of the two-photon microscope. Tissue in which fluorescent proteins were expressed was often fixed and immunostained (see above), and subsequently imaged on a confocal microscope  in the Leduc Imaging Facility, Brown University. Confocal and two-photon stacks were processed in Fiji (49. The laser power used (910 nm) was ~3%. The frame rate was 15 Hz. For imaging responses in dendrites, 128 × 256 pixel fields of view were used with a zoom of 4.5× or 7× (56 × 113 or 36 × 73 µm2 FOVs). Light stimulus presentation was synchronized to the fly-back times in the scanning of the microscope so that they did not interfere with the measured signal.Imaging of calcium indicator signals were acquired using the two-photon microscope and conditions described above, as has been done previously0, we averaged the brightness over 0.5 s before every stimulus presentation. ROIs were chosen for analysis if their time averaged responses surpassed a threshold ΔF/F0 (0.3–0.6) during at least 3 or 6 stimulus presentations out of 24. ROIs that had exceptionally noisy responses, or that seemed in the projection image to belong to RGC dendrites rather than the VGluT3 plexus were discarded. In Figs. 51, and ROIs were considered orientation or direction selective if the corresponding index exceeded 0.2. In Fig. Functional imaging analysis was done using Fiji see above) and custom written Matlab routines. For each movie, a standard deviation projection was made, over which ROIs were manually marked over brighter dendrites Fig. , and traove and cMice were anaesthetized with isoflurane . A viral vector inducing Cre-dependent expression of a payload (see below) was injected into the vitreous humor of the right eye through a glass pipette using a microinjector . Analgesia  was applied to the eye ~2 min before the injection, and immediately following the injection  to minimize postoperative pain. Mice were then taken off anesthesia, recovered within several minutes, and monitored for 48 h following the procedure. Animals were killed and retinas removed 14–21 d later.pAAV2/2-hSyn-DIO-hM4D(Gi)-mCherry  was injected intraocularly in VGluT3-Cre mice, causing Cre-dependent expression of hM4D(Gi), a modified human muscarinic M4 receptor, that is an inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD). AAV2/2-EF1a-DIO-hChR2(H134R)-EYFP  was injected VGluT3-Cre mice to express ChR2 in a Cre-dependent manner, but mostly caused expression in too few of the VGluT3 ACs to drive optogenetic responses in postsynaptic RGCs  and introduced through the microscope objective and GFP excitation filter cube, resulting in a spectrum peak at 480 ± 10 nm, and illumination over an area of 1 mm in diameter. The light intensity at the sample was 0.9–1.4 nW/µm25 was used. The imaged volume dimensions were 50 × 210 × 260 µm3 with the short dimension spanning the IPL and parts of the GCL and INL layers of the retina. The pixel size was 13.2 nm2 and the section thickness 26 nm. The images contained intracellular details, e.g., synaptic vesicles. The webKnossos platform  was used for tracing neuronal skeletons and annotating synapses. For the plot of stratification depth , ‘k0725’Averaging, error estimation and the statistical tests used are detailed in the “Methods” sections above. No adjustments were made to p values. Independent repetitions: each cell in the following data was recorded in a different retina and mouse: Figs. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Our study reveals that the electronic properties of penta-SiC2 and g-SiC3 nanoribbons are closely related to their width and orientation. Specifically, one type of penta-SiC2 nanoribbons exhibits antiferromagnetic semiconductor characteristics, two types of penta-SiC2 nanoribbons have moderate band gaps, and the band gap of armchair g-SiC3 nanoribbons oscillates in three dimensions with the width of the nanoribbon. Notably, zigzag g-SiC3 nanoribbons exhibit excellent conductivity, high theoretical capacity (1421 mA h g−1), moderate open circuit voltage (0.27 V), and low diffusion barriers (0.09 eV), making them a promising candidate for high storage capacity electrode material in lithium-ion batteries. Our analysis provides a theoretical basis for exploring the potential of these nanoribbons in electronic and optoelectronic devices as well as high-performance batteries.The dimensions of nanoribbons have a significant impact on their material properties. In the fields of optoelectronics and spintronics, one-dimensional nanoribbons exhibit distinct advantages due to their low-dimensional and quantum restrictions. Novel structures can be formed by combining silicon and carbon at different stoichiometric ratios. Using density functional theory, we thoroughly explored the electronic structure properties of two kinds of silicon–carbon nanoribbons (penta-SiC The pz orbitals perpendicular to the graphene plane form energy bands with linear dispersion relations near the Fermi level, exhibiting the characteristics of a Dirac cone, which endows graphene with excellent electrical and thermal conductivity properties [2 hybridization in carbon atoms, graphene is currently the most mechanically strong 2D material [2 (graphene), sp3 (diamond), and sp (ethylene). Although silicon and carbon are in the same column of the periodic table, their bonding characteristics are markedly different. Silicene, a 2D honeycomb lattice made up of sp2-sp3 hybridization, possesses several superior properties and good compatibility in devices that make it a promising substitute for graphene. Currently, silicon is the main component of semiconductor electronic products. However, both graphene and silicene exhibit zero band gaps, which limits their widespread use in optoelectronic devices such as LEDs, field-effect transistors, and solar cells. Searching for new 2D materials that surpass traditional materials will have significant implications for future applications. For instance, combining silicon and carbon to form 2D binary compounds could lead to novel material properties. Different stoichiometric ratios of carbon and silicon generate novel 2D layered structures, such as SiC [2 (penta-SiC2) [2 siligraphene (g-SiC2) [2 silagraphene [3 siligraphene (g-SiC3) [7 siligraphene (g-SiC7) [Since the discovery of graphene in 2004, the two-dimensional (2D) layered structure has attracted significant scientific interest . Graphenoperties . Due to h as SiC , folded ta-SiC2) , SiC2 si(g-SiC2) , SiC2 sigraphene , SiC3 si(g-SiC3) , and SiC(g-SiC7) . These s(g-SiC7) ,12,13,14Science about the topological localized excitons found in single armchair graphene nanoribbons. The findings of their research offer a nanoscale solution for schemes that incorporate the electron, magnetic, and photon degrees of freedom in quantum mechanics [2 and g-SiC3 nanoribbons, we explored the electronic structures of penta-SiC2 and g-SiC3 nanoribbons. Considering the electrical conductivity of zigzag g-SiC3 nanoribbons, we explore the potential of zigzag g-SiC3 nanoribbons as anode electrode materials for LISs. Similar materials have been reported experimentally, such as the application of Si@SiC composite and graphene nanoribbons in the field of lithium-ion batteries [The properties of materials are closely related to their dimensions. One-dimensional (1D) nanostructures, such as nanoribbons, nanotubes, and nanowires, display properties beyond those of 2D systems because of their low dimensionality and quantum confinement. They exhibit electronic bandgap changes associated with quantum confinement and edge effects, as well as local spin-polarized edge states with specific edge geometries. This makes 1D nanostructures promising candidates for spintronics, optoelectronics, and other applications that require tailored electronic and magnetic properties and has attracted widespread attention over the past two decades. For example, graphene nanoribbons (GNRs) exhibit unique properties that are distinct from those of bulk graphene when cut into smaller widths ,16,17. Zechanics . Differeatteries ,24,25. Fatteries . A compr2 and g-SiC3 nanoribbons, with varying widths, growth directions, and edge environments. The results revealed that the electronic properties of these nanoribbons were strongly influenced by their orientation and width. Specifically, the a-type penta-SiC2 nanoribbon exhibited characteristics of an antiferromagnetic semiconductor, which could be beneficial for the development of next-generation optoelectronic devices. Meanwhile, the bandgap of armchair g-SiC3 nanoribbons exhibited a 3D oscillation pattern with increasing width. It is worth noting that zigzag g-SiC3 nanoribbons show promise as a potential electrode material for lithium-ion batteries because of their excellent conductivity, a theoretical capacity of 1421 mA h g−1, the moderate open circuit voltage, and low diffusion barriers. By systematically studying the electronic properties of these nanoribbons with different widths and orientations, we can identify the desired material properties and promote the application of such materials in the field of optoelectronics and batteries.Using first-principles calculations, we investigated the electronic properties of two types of silicon carbide nanoribbons, namely penta-SiC2 and g-SiC3 nanoribbons were calculated using the hybrid Heyd–Scuseria–Ernzerhof (HSE06) functional [-x direction, while a 15 Å vacuum layer is applied in the y− and z− directions. The Brillouin zone was sampled using a 2 and g-SiC3, while for the nanoribbon model, a bE) using the formula This study employs first-principles calculations based on density functional theory using the VASP (Vienna ab initio simulation) software package to calculate relevant geometric structures, electronic structures, and energies . The elenctional . Plane w adopted . We conste tools ,34. The 2, where each unit cell consists of two Si atoms and four C atoms. The optimized Si-C bond length is 1.908 Å, and the C-C bond length is 1.362 Å. According to the electronic band structure in 2 is an indirect bandgap semiconductor with a bandgap of 1.38 eV and 2.49 eV at PBE and HSE06 levels, respectively. The results agreed well with the previous literature [2 is 7.297 eV/atom, which is lower than that of graphene (9.176 eV/atom) but higher than that of g-SiC2 siligraphene (6.46 eV/atom) [3 siligraphene belongs to the hexagonal space group P6/mmm, as shown in 3 is a hybrid of Si and C atoms, exhibiting characteristics similar to graphene. The C–C bond (1.439 Å) shows the characteristic of weak C=C double bonding because it is slightly longer than that of graphene (1.42 Å) but shorter than the C–C single bond in ethane (1.54 Å). The Bader analysis shows that electrons are transferred from Si to C. The electronic band structure  but higher than that of g-SiC2 siligraphene (6.46 eV/atom) [2 hybridization, Si atoms prefer to adopt sp2-sp3 hybridization and form the buckled structure of silicene, making the stability of planar silicene decrease as the number of Si atoms increases.The g-SiCtructure d reveals2 and g-SiC3 nanoribbons, taking into account their unique properties as one-dimensional nanostructures. Due to their low dimensions and quantum confinement effects, nanoribbons demonstrate superior functionality when compared to 2D systems. Specifically, nanoribbons exhibit modifications in electronic bandgaps associated with quantum confinement and edge effects, as well as local spin-polarized edge states specific to the edge geometry. Considering the aforementioned qualities, we seek to better understand the electronic structure of penta-SiC2 and g-SiC3 nanoribbons.This study investigates the electronic structural characteristics of penta-SiC2 nanoribbons were analyzed to gain insight into their behavior under different conditions first. Four types of nanoribbon structures were examined while taking into account different directions and end atoms. The nanoribbon’s width was defined as the number of edge silicon atoms with each edge saturated with hydrogen atoms. The penta-SiC2 nanoribbons maintained their folding characteristics. In the subsequent sections, we will discuss the properties of these four types of nanoribbons in the order presented in 2 nanoribbons in different conditions.The electronic properties of penta-SiC2, are made up of carbon atoms located at the edges. We investigated the spin configuration of these nanoribbons and found that the antiferromagnetic configuration (AFM) is the most stable, with magnetic moments mainly concentrated on the edge carbon atoms, as shown in y orbital of carbon, while the lowest unoccupied state at points Г is contributed by the pz orbital of the edge carbon. The bandgap of nanoribbons increases with the increase in the nanoribbon’s width. These characteristics of the nanoribbon can be useful for the development of a new generation of optoelectronic devices.The first type of nanoribbons, called a-SiC2, has edge atoms composed of Si and C. Our research has focused on investigating the possible spin configurations of the second type of nanoribbons and we have obtained two configurations  differs from the previous three types in direction, which are composed of silicon and carbon atoms that have been saturated with hydrogen atoms. In this study, we provide a comprehensive analysis of the band structure and density of states for a nanoribbon with a width of 10 (w = 10). This type of nanoribbon is a nonmagnetic indirect bandgap semiconductor .We explored the electronic properties of g-SiCThe calculations have shown that this nanoribbon is a direct bandgap semiconductor with the gap located at Г point from the energy-band structure in 3 nanoribbons with a width of seven (w = 7) as anode electrode materials for LISs. Our analysis reveals that nanoribbons of different widths exhibit metallic behavior. The electronic band structure for both PBE and HSE06 methods of the w = 7 nanoribbon shows that both spin-up and spin-down orbitals cross the Fermi level, indicating metallic behavior . Li storage capacity. To evaluate the average open circuit voltage (aveV), we consider a typical half-cell reaction against Li/Li+: aveV by computing the total energy difference between Li-intercalated SiC3 nanoribbons with different Li concentrations using the definition [The performance of LIBs is affected by two important parameters: the average electrode potential and the finition :Vave=−ELThe resulting electrode potential ranges from 0.10 to 0.45 V, with an average value of 0.27 eV, as evidenced by xmax represents the maximum fraction of Li in g-SiC3 nanoribbon, F is the Faraday constant (26.8 A h mol−1), and 3 nanoribbon (452.7 g mol−1). The theoretical capacity of g-SiC3 nanoribbon, estimated to be 1421 mA h g−1 using the aforementioned formula, is approximately 3.8 times higher than that of commercial graphite (372 mA h g−1) [−1) [2C (526 mA h g−1) [2N4 (547 mA h g−1) [−1) [−1. This highlights the potential of g-SiC3 nanoribbon as a high-capacity LIB anode material. The remarkable capacity can be ascribed to the unique arrangement of Si and C atoms. Overall, the promising features of g-SiC3 nanoribbon make it an attractive candidate for high-capacity LIB.The maximum theoretical capacities of Li atoms can be determined using −1) [−1) , Mo2C (5A h g−1) , NbGe2N4A h g−1) , and dum−1) [−1) . Despite3 nanoribbons to those in graphene (0.37 eV) [3 nanoribbons, which implies high charging and discharging rates.Using the CNEB strategy, we determined the energy profile of diffusion for a Li atom between two steady states. Specifically, we considered the diffusion path 0.37 eV) , TiF3 (00.37 eV) , silicon0.37 eV) , and pho0.37 eV) , we obse3 nanoribbons. Our findings demonstrate that this nanoribbon offers an exceptionally high capacity (1421 mA h g−1), coupled with a low potential barrier (0.09 eV) and moderate average electrode potential (0.27 V), all of which together indicate that it has significant potential as an anode electrode material.Based on the calculations presented in this section, we propose a high-capacity anode material for LIBs: the zigzag g-SiC2 nanoribbons and two types of g-SiC3 nanoribbons with different widths. Our results suggest that the band structures of the nanoribbons are closely related to their widths and growth directions. Notably, type-a penta-SiC2 nanoribbons exhibited antiferromagnetic behavior and a-SiC2 and b-SiC2 had moderate band gaps, which make them suitable for applications in optoelectronic devices. Additionally, the armchair g-SiC3 nanoribbons showed three-dimensional oscillation of the band gap with width increase, whereas the zigzag g-SiC3 nanoribbons possessed metallic properties. It is worth highlighting that the zigzag nanoribbons hold potential as high-capacity anode electrode materials for LISs, displaying a high capacity of 1421 mA h g−1, a moderate open-circuit voltage of 0.27 V, and a low diffusion barrier of 0.09 eV. Our systematic investigation of the electronic structures of penta-SiC2 and g-SiC3 nanoribbons with varying widths and directions could contribute to finding the desired material properties needed to advance their applications in fields such as optoelectronics and battery technology.In this study, we utilized the first-principles method to investigate the electronic structures of four types of penta-SiC


Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues  Protein synthesis is a highly regulated process involving the precise orchestration of many chaperones, co-factors, enzymes, and biomolecular building blocks. It is critical at every level of the life cycle, from development through aging, and is central to stress adaptation. Protein synthesis largely determines the folding load on the proteostasis network, which regulates protein production, folding, trafficking, and degradation to maintain a functional proteome. The imbalance of the proteostasis network caused by age-associated stress or acute environmental insults leads to misfolding of proteins, accumulation of aggregates, and eventually to disease .Caenorhabditis elegans identified several ribosomal subunits whose knockdown increased the aggregation of polyglutamine (PolyQ) proteins  and translation elongation (TE) inhibitors by screening a series of translation inhibitors for their ability to lower protein synthesis in t manner . To monit manner . In thist manner . The sixt manner . Based ot manner .C. elegans, we generated a bi-cistronic reporter to co-express both GFP and tdTomato under the control of the myo-3 promotor and an internal ribosomal entry site (IRES)  . In tranOn the other hand, the TI inhibitors 4E1RCat and salubrinal only decreased the fluorescence of the green channel, consistent with an initiation-dependent translation of the GFP mRNA sequence and the initiation-independent translation of the IRES-tdTomato sequence . HoweverC. elegans. Using the SUnSET assay and our bi-cistronic reporter, we show that cycloheximide directly reduces the concentration of newly synthesized proteins  mutants. Only the TE inhibitors anisomycin and lycorine protected hsf-1(sy441) from HS-induced proteotoxicity, while the TI inhibitors 4E1RCat and salubrinal did not  controlled by the transcription factor HSF-1 to protect from heat stress (HS). Previous work by us and others resulted in contradictory findings on whether protection from heat by translation inhibition depends on HSF-1 . Therefo did not . These rTo broadly confirm the ability of TE inhibitors to protect from thermal proteotoxicity through improved proteostasis, we conducted sequential detergent extractions to biochemically isolate and quantify soluble and insoluble proteins in wild-type (N2) animals . FollowiTE inhibitors are reported to free up folding capacity by reducing overall protein synthesis. To monitor folding capacity in live imaging, we developed an experimental procedure to examine how translation inhibition will dynamically affect protein aggregation in real time in the PolyQ::YFP strain AM140 . The AM1Only TE inhibitors lycorine and anisomycin significantly reduced HS-induced PolyQ aggregation, while the TI inhibitors 4E1RCat had a small effect and salubrinal had no effect . Pre-treThe data thus far suggest that inhibition of translation protects from folding stress by an HSF-1-dependent and -independent mechanism determined by the inhibited translation step. The proteasomal system is another critical proteostasis mechanism by which cells clear protein aggregates. The proteasomal system ubiquitinylates misfolded proteins by ubiquitin ligases to target them for degradation by the 26S proteasome. Blocking proteasome degradation by bortezomib, a specific inhibitor of the 20S subunit, results in the formation of protein aggregates and proteotoxic stress .hsf-1(sy441) L4 animals with either TI or TE inhibitors for 12 hr, followed by the addition of bortezomib, and measured survival on day 8 of adulthood (hsf-1(sy441) background because it serves as a proxy of reduced folding capacity given the defective HSR, allowing us to further investigate the relationship between reduced translation and its conditional dependency on hsf-1. Compared to non-treated controls, bortezomib decreased the survival of both genotypes. TE inhibitors improved the survival of the hsf-1(sy441) animal back to untreated levels but only showed a non-significant tendency to improve the survival of N2 wild-type animals (hsf-1(sy441) animals to shrink in size (sma phenotype)  . We quanmal size . A similsma phenotype was specific to hsf-1(sy441) animals or if the combination of proteasome toxicity with impaired protein folding capacity caused it. We, therefore, treated PolyQ35::YFP transgenic animals for 12 hr with the four different translation inhibitors, followed by bortezomib. As expected, treating PolyQ::YFP animals with bortezomib caused extensive protein aggregation. As before, the inhibition of TI exacerbated bortezomib toxicity and was not further quantified. As with hsf-1(sy441), bortezomib treatment also induced the sma phenotype in the PolyQ35::YFP transgenic animals, which was almost entirely rescued by anisomycin (We next asked if the induction of the isomycin .hsf-1(sy441) animals. TE inhibitors did not seem to affect proteasome activity in either strain. In contrast, TI inhibitors reduced N2 proteasome activity compared to DMSO-treated controls, providing a rationale for their increased toxicity in our proteasome survival assay (hsf-1(sy441) mutants showed significantly decreased proteasome activity compared to N2 controls, which was not further reduced by TI inhibitors in hsf-1(sy441).We then directly tested the effect of each inhibitor on proteasome activity in both N2 and al assay . hsf-1 animals (hsf-1(sy441) mutants. As expected, both 4E1RCat and salubrinal failed to extend lifespan in hsf-1(sy441) mutants significantly. This result is consistent with genetic work by both the Rodgers and Tavernarakis labs, which showed inhibition of TI extends lifespan dependent on HSF-1   .hsf-1(sy441) mutants by ~20% (hsf-1(sy441) mutants, as the increase in lifespan did not reach the lifespan of wild-type animals. Taken together, however, our data demonstrate that longevity induced by translation inhibition, whether through targeting TE or TI, subsumes several different mechanisms that lead to longevity.As  by ~20% . Similar by ~20% . We inteWe next tested the ability of all four inhibitors to reduce age-associated protein aggregation. If inhibition of translation alone is sufficient to reduce protein aggregation independently of any downstream mechanisms, all four inhibitors should reduce age-associated protein aggregation. Conversely, if the reduction of age-associated protein aggregation is closely linked to longevity, then only the TI inhibitors should reduce protein aggregation. We treated day 1, N2 animals with 100 μΜ of each of the four inhibitors and allowed the animals to age for 8 days, after which we separated proteins based on solubility . We founreduced folding load model without generating additional protein folding capacity. Separately, inhibition of TI protects and improves longevity by the selective translation model that depends on HSF-1.Taken together, our data suggest that inhibition of TE rescues proteostasis-compromised animals by the The canonical model that explains the requirement of HSF-1 to induce longevity by inhibiting TI suggests that blocking TI activates the HSR via HSF-1 that then initiates the transcription of HSP chaperones to increase folding capacity . In conthsf-1(sy441) animals with the four inhibitors and measured the resulting concentration of newly synthesized proteins. As before, all four inhibitors reduced the concentration of newly synthesized proteins in N2. In contrast, TI inhibitors increased the concentration of newly synthesized proteins by 1.5- to 2-fold in the hsf-1(sy441) animals  directly contributes to the proper function of the TI machinery. We treated N2 or  animals . We conf animals . Further6.2::GFP .ifg-1, by RNAi in N2 and hsf-1(sy441) animals and measured the concentration of newly synthesized proteins. As seen for 4E1RCat, the knockdown of eIF4G/ifg-1 reduced the protein concentration in N2 but dramatically increased it in hsf-1(sy441) mutants  mutants which carry a splicing defect that results in an overall reduction of eIF4G/ifg-1 protein (ifg-1(cxTi9279) animals  mutants as inhibition of eIF4G/ifg-1 fails to lower protein concentrations in this strain.To further confirm that the lifespan extension of 4E1RCat is caused by on-target action, we measured lifespan extension in N2 and  protein . As we p protein  4E1RCat  protein . However animals , showingA substantial body of genetic and biochemical work suggests that lowering the concentration of newly synthesized proteins reduces the load on the proteostasis machinery. We asked if these effects could be pharmacologically replicated to exploit them for therapeutic purposes in disease models .C. elegans. We intended to compare the selective translation model, in which the selective reduction of specific proteins and the selective translation of others protect from proteotoxicity to the reduced folding load model, in which the non-specific reduction of the concentration of newly synthesized proteins is protective.We set out to study how pharmacological inhibition of TI or TE improves proteostasis and increases longevity in adult We compared the non-selective lowering of protein concentration by TE inhibitors to the selective lowering of initiation-dependent proteins by TI inhibitors. Our studies reveal that pre-existing proteotoxicity, as might be observed in a disease state, dictates the mode of translation inhibition necessary to restore proteostasis. As a result, the mode of translation inhibition, either inhibiting TI or TE, results in distinct and sometimes opposing outcomes for the health of the animals.The most striking dichotomy was seen when pre-existing proteotoxicity—caused by the lack of either HSF-1 or the expression of the aggregation-prone PolyQ protein—was combined with the inhibition of the proteasome. These combined insults led to increased protein aggregation, shrinking body size, and death of the animals . Inhibithsf-1(sy441) mutant animals, with each mode of inhibition only able to extend the lifespan in one of the strains. Overall, TE inhibition effectively rescued proteostasis in animals with pre-existing proteotoxicities, such as a compromised HSR, reduced proteasome activity, or the expression of aggregation-prone proteins. Conversely, inhibition of TI did little to rescue pre-existing proteotoxicity or worsen it but proved effective at preventing emerging proteotoxicity, for example, with age. Notably, both anisomycin and cycloheximide have previously been reported to extend lifespan in wild type animals  mutants and the inability to extend their lifespan shows that lowering the concentration of newly synthesized proteins is necessary for the beneficial effects. On the other hand, the finding that TE inhibitors protect from proteotoxic stress but do not extend lifespan shows that lowering the concentration of newly synthesized proteins is sufficient to protect from proteotoxic stress but is not sufficient to extend lifespan in wild-type, which appears to require selective translation.The inability of TI inhibitors to reduce the concentration of newly synthesized proteins in ifg-1  may influence the concentration of newly synthesized proteins by either modulating protein degradation or synthesis. Our data show that proteasome activity and, by extension, degradation are lowered by both inhibition of TI and lack of HSF-1. Hence, combining both may indirectly increase the concentration of newly synthesized proteins through reduced degradation. However, inhibition of TI did not further decrease the already low proteasome activity in HSF-1 mutants . Furtherhsf-1(sy441) mutant, the TI machinery still prevents uncontrolled translation but less effectively, leading to a slight but detectable increase in newly synthesized proteins in HSF-1 mutants  cooperates with TI factors to form a checkpoint that prevents random unregulated translation. The TI factors are part of the checkpoint but also initiate regulated translation by giving ribosomes access to mRNA. Thus, when TI factors are inhibited or knocked down, HSF-1 or one of its targets maintains the checkpoint preventing uncontrolled translation. Without any TI activity, maintaining the checkpoint reduces protein synthesis. In the  mutants . Once thBoth the degradation and the cooperative checkpoint model explain the observed results but need to invoke a speculative connection between HSF-1 to either TI or protein degradation. Our results unexpectantly couple the ability of the TI machinery to control the concentration of newly synthesized proteins to HSF-1, thereby explaining its requirement. However, our results do not provide a mechanism by which HSF-1 couples the concentration of newly synthesized proteins to TI.Michael Petrascheck is the Lead Contact and may be contacted at pscheck@scripps.edu. This study did not generate new unique reagents; however, the natural product Amicoumacin C was obtained as a gift from Dr Shigefumi Kuwahara, Ph.D. (Tohoku University). This reagent is not available without total chemical synthesis.hsp-16.2p::GFP+rol-6(su1006)]], AM140 [rmIs132[Punc-54::q35::yfp]], KX54 [ifg-1(cxTi9279)] and PS3551 [hsf-1(sy441)].The Bristol strain (N2) was used as the wild-type strain. In addition, the following worm strains used in this study were obtained from the Caenorhabditis Genetics Center : CL2070  animals were prepared as above in 6 cm culture plates and treated with water or 100 μM lycorine/anisomycin on day 1. On day 4, 25–35 animals were transferred to 6 cm NGM plates in triplicate for each condition and were transferred to the non-permissive temperature of 36°C. Every hour, survival was scored by lightly touching animals with a worm pick and scoring for movement.Age-synchronized N2 or PS3551 ) was added, and animals homogenized mechanically. An aliquot of this total lysate was saved. In an ultracentrifuge tube, two volumes of SDS Extraction buffer  were added to 1 volume of total lysate and was centrifuged at 20,000 × g for 30 min. The extraction was repeated two times to remove all SDS-soluble proteins. The remaining insoluble pellet was suspended briefly in 20 µL urea buffer  and sonicated. 18 µL of the insoluble suspension was added to 6 µL 4× Laemmli buffer  supplemented with 10% 2-mercaptoethanol  and boiled for 5 min, then directly loaded onto SDS-PAGE gel . Gels were stained with Sypro Ruby according to the manufacturer’s directions. Gels were quantified in ImageJ by dividing the integrated intensity of each full insoluble lane by the integrated intensity of the corresponding full total protein lane after subtracting a similar area background lane, then normalizing to the DMSO control.10,000 N2 worms were sorted into a 15 cm liquid culture dish using the COPAS Biosorter (Union Biometrica). For heat shock-induced aggregation experiments, worms were treated with either DMSO or anisomycin (100 μΜ) for 12 hr on day 1 of adulthood, then subjected to a 2 hr heat shock at 36°C. After 12 hr of recovery, the animals were washed three times with S-complete buffer, once with PBS, and then flash-frozen in liquid nitrogen. 500 µL of cold lysis buffer  or day 5 for PolyQ using the 10× objective with the ImageXpress Micro XL and Metaexpress microscopy software.Animals were prepared as above in 6 cm liquid culture dishes. At the late L4 stage, animals were pre-treated with DMSO or 100 μΜ anisomycin. After 12 hr, the animals were treated with 75 μΜ bortezomib. Body length was measured on day 3 for The Chemicon 20S Proteasome Activity Assay Kit (Cat. No APT280) was used according to the manufacturer’s instructions. In short, 10,000 age-synchronized animals were grown in liquid culture on 10 cm plates. DMSO or inhibitors were added on day 1. On day 5, animals were collected and washed 3× with cold DPBS, then once with 1× assay buffer. Worms were flash-frozen in liquid nitrogen. After three biological replicates were harvested in this way, the frozen animals were broken open with a beak mill homogenizer (Fisherbrand). Protein concentrations were determined by the Bradford protein assay. 200 μg of sample was loaded with assay mixture into a 96-well plate and incubated for 1 hr at 37°C. Fluorescence was measured on the Tecan Safire II with a 380/460 nm filter set. Positive control: 5 µL of 20S proteasome positive control (Chemicon Part No. 90205). Negative control: N2 lysate treated with 25 mM lactacystin, a 20S proteasome inhibitor (Chemicon Part No. 90208).hsp-16.2p::GFP+rol-6(su1006)]] or AM140 [rmIs132[Punc-54::q35::yfp]] age-synchronized animals were treated with DMSO or 100 μΜ drug at late L4. 4–12 hr later, on day 1 of adulthood, 1.5 mL of the treated animals were transferred from liquid culture into an Eppendorf tube, washed twice with S-complete, pelleted, and then transferred to 6 cm NGM plates using S-complete. Once the animals were completely dry on the NGM plate, they were transferred to a 36°C incubator, plates upside down, for 1–2 hr.CL2070 [dvIs70 [Animals were washed from NGM plates using 0.2% HHPPA (2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone) (CAS[106797-53-9]) dissolved in S-complete into a 2 mL Eppendorf tube. Animals were washed twice with 0.2% HHPPA, then suspended in 0.3 mL 0.2% HHPPA. 2.5 µL of this solution was seeded into a single well of a 384-well plate containing 2.5 µL 30% PEG-DA  in S-complete. After 5 min, to allow the solutions to diffuse, the animals were immobilized by subjecting the 384-well plate UV light using a routine laboratory gel viewer  for 30 s. 45 µL S-complete buffer was added on top to prevent desiccation. In general, each well contained 5–10 worms.Time-lapse brightfield and fluorescence images were taken with a 10× objective using the ImageXpress Micro XL over 15 hr. The number of PolyQ aggregates, or total YFP fluorescence, in the whole worm, was determined by analyzing images using a custom pipeline created in CellProfiler.C. elegans were prepared in a liquid medium, as described above, and seeded into flat-bottom, optically clear 96-well plates  containing 150 µL total volume per well, as previously described  was measured for each full lane using ImageJ. A similar-sized band with no signal was used to subtract the background, and then each intensity normalized to its corresponding GAPDH loading control. Finally, each band’s integrated intensity was normalized to the wild-type DMSO control for quantification and statistics. Significance was determined by one-way ANOVA with Šídák multiple comparisons test.The number of n represents the total number of animals over the three individual replicates shown as the average percentage survival and SEM, calculated using GraphPad Prism. Significance was determined by using a row-matched two-way ANOVA with Šídák multiple comparisons test.The number of n represents the total number of animals whose length was measured in one experiment. Depicted are the mean and standard deviation calculated using GraphPad Prism. Significance was determined by the two-tailed unpaired t-test. Similar results were observed across three independent experiments.Survival was scored manually by visually monitoring worm movement using an inverted microscope thrice weekly. Statistical analysis was performed using the Mantel-Haenzel version of the log-rank test as outlined in https://cellprofiler.org/.The software used in this study (Cell Profiler) is available at  Inhibition of translation has been found as a conserved intervention to extend lifespan across a number of species. In this work, the authors systematically investigate the similarities and differences from pharmacological inhibition of protein synthesis at the initiation or elongation steps on longevity and stress resistance. These experiments are important for conceptualizing how translation inhibition actually extends lifespan and promotes proteostasis. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Pankaj Kapahi, and James Manley as the Senior Editor. The following individual involved in review of your submission have agreed to reveal their identity: Brian M Zid (Reviewer #1).Thank you for submitting your article "Proteostasis is differentially modulated by inhibition of translation initiation or elongation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.eLife and is thus not suitable for publication in eLife in its current form. The Reviewing Editor would be glad to discuss beforehand it makes more sense to consider revision for eLife or whether you may want to consider submitting the work elsewhere.After consultation with the reviewers, and based on the detailed review below, we have decided that the presented work needs significant revisions to be considered for publication by Reviewer #1 (Recommendations for the authors):Do the authors believe that initiation inhibitors don't slow protein production in hsf-1 mutants? If so, this should be shown. Do the authors believe there is a toxic side effect from initiation inhibition in hsf-1 mutants which masks the benefit from reducing protein synthesis in this background? Either way they should discuss this and revise their conclusion for elongation inhibition, similarly to what they have done for initiation inhibition.One potential alternative explanation for the effect of elongation inhibition in hsf-1 and on proteostasis is that as reduced elongation speed is important for nascent protein folding  and coding fidelity , that low doses of elongation inhibitor slow translation elongation speed leading to enhanced proteostasis, outside of just the changes in overall protein synthesis.Reviewer #2 (Recommendations for the authors):C. elegans community is very useful. However, the effect of the compounds used in this study should be clearly shown in C. elegans, as the whole manuscript relies on the comparison of translation inhibition at selected steps. The effectivity of the drugs to inhibit translation initiation and elongation, respectively, was shown using puromycin incorporation , which measures changes in overall protein synthesis and not changes in translation rates at selected steps. The analyzed compounds were studied in other systems such as mammalian cells and yeast, however, the source given in this manuscript does not show if they were previously used and validated in C. elegans. To make the claim more solid that these drugs do inhibit specific steps of translation in the nematode, the authors should have used additional ways to show how the compounds act on the translation process, such as polysome profiling. For example the proposed translation initiation inhibitor salubrinal is suggested to inhibit eIF2alpha phosphatases (hence inducing ISR and reducing translation initiation). However, to my best knowledge, its mechanism of action is unknown and, more importantly, the eIF2alpha phosphatases of C. elegans are not identified, making it speculative if salubrinal does inhibit initiation. Also, while both anisomycin and lycorine are supposed to inhibit elongation and were shown to inhibit overall translation to similar extends , the strength of their effect on survival during heat shock is different between the drugs , raising the question if they indeed act similarly on translation or if they might have off-target effects. For example, anisomycin is discussed to have strong side effects (DOI: 10.1128/MCB.15.9.4930).1) Validation of the drugs. Using different compounds to inhibit translation at different states is an interesting approach and having these compounds validated for the 2) Consistency of compounds and genotypes used throughout manuscript. Comparing the effects of the inhibition of different translation steps during selected external stresses and in different genetic models is a great approach. Unfortunately, because of the inconsistent use of genotype-treatment-combinations, the lack of Wildtype controls in some assays and splitting graphs based for example on treatment types, it is hard to interpret the results in a global way and to truly compare between initiation and elongation inhibition.3) Statement that elongation inhibitors reduce ongoing toxicity by reducing folding load independently of hsf-1. Some contradictions in the effect of elongation inhibition are not addressed. Elongation inhibition is discussed to reduce toxicity by reducing protein folding load. While it is shown that inhibition of elongation reduces protein aggregation upon heat shock  and in a PolyQ model , protein aggregation is not lowered by elongation inhibition in aged worms . It is further claimed that elongation inhibition acts independently of hsf-1 in many of the observed phenotypes, but sometimes Wildtype controls are missing to rule out a partial dependency. Furthermore, there could be other factors than hsf-1 involved that are changed upon elongation inhibition and mediate the phenotypes instead of (or in addition to) the overall reduction of protein and hence the increase in folding capacity. Also the inhibition of initiation might reduce folding load . More direct data to compare protein aggregation upon inhibition of initiation with elongation during stresses are mainly missing. Translation itself is not compared on a molecular level between the different translation modulation modes.4) Effects on lifespan. The lifespan assays in Figure 5 are very interesting and underline the previous observations nicely, as they directly compare two initiation and elongation inhibitors in Wildtype and hsp-1 mutant contexts. With regards to elongation inhibition, there might be contradictions to other published data that found inhibition of elongation using cycloheximide to increase lifespan (DOI: 10.1038/srep18722). One more reason why it would be great to have the effect of the elongation inhibitors on translation used here validated.5) Given the high focus of the manuscript on the difference between the inhibition of translation initiation and elongation, translation itself could have been further analyzed using the different compounds and during selected stresses (for example polysome profiling or ribo seq). The authors claim that their results are consistent with a model proposed by Rogers et al. (2011), saying that lifespan extension through inhibition of translation initiation acts via translation of selected factors. Could the status of some of these factors be checked upon translation inhibitor treatment to verify this?6) Figure 2D,E; Figure 4C; Figure 5C,D,E pose multiple questions that remain unanswered: What is the difference between age-associated protein misfolding on the one hand and proteotoxicity caused by heat, proteasome dysfunction, and PolyQ stretches on the other hand, and why do animals react differently to it when treated with initiation or elongation inhibitors? Why does inhibition of initiation protect from age-associated protein toxicity but not from proteasome-misfunction-induced protein toxicity? Why does inhibition of elongation protect from proteasome-misfunction-induced protein toxicity but not from age-associated protein aggregation?Reviewer #3 (Recommendations for the authors):hsf-1's role in EIs but not IIs protecting against proteasomal dysfunction: the data is Figure 3 make this a weak conclusion at best. In the paradigm used, the difference between WT and hsf-1 mutant survival is only 20% and the 'limited protection' vs. 'highly protective effects' of the respective chemical classes are not apparent. From the figure, the impact seems to be similar on both genotypes. The 'morphology' improvements brought about by EIs are not explained and the images in 3G cannot be used to measure 'unc' phenotypes.In Figures 2B, C, the data for concluding hsf-1 dependence/independence of IIs and EIs thermotolerance is weak. The graphs look moderately different from each other but only one P value is provided (does it apply to both treatments? what is the comparison?) that is not very convincing (P 0.05 vs. 0.02). No details for means/SEM/number of animals and trials are provided either.Data for lifespan  and thermotolerance  survival assays are from one trial only and do not include details . Additional independent trials are critical for verification.How were the protein quantifications performed for the Puromycin immunohistochemistry (1A) and the insoluble/soluble fractions . Were specific regions or bands' intensity measured or the whole lane?Is the inhibitor treatment for 72h (3 days) or 4 days? The article mentions 72h  but figures  show 4 days. Reviewer #1 (Recommendations for the authors):Do the authors believe that initiation inhibitors don't slow protein production in hsf-1 mutants? If so this should be shown. Do the authors believe there is a toxic side effect from initiation inhibition in hsf-1 mutants which masks the benefit from reducing protein synthesis in this background? Either way they should discuss this and revise their conclusion for elongation inhibition, similarly to what they have done for initiation inhibition.One potential alternative explanation for the effect of elongation inhibition in hsf-1 and on proteostasis is that as reduced elongation speed is important for nascent protein folding  and coding fidelity , that low doses of elongation inhibitor slow translation elongation speed leading to enhanced proteostasis, outside of just the changes in overall protein synthesis.Thank you for this point. As mentioned above and shown in the new Figure 6, initiation inhibitors do not reduce the concentration of newly synthesized proteins in hsf-1(sy441). On the contrary, they surprisingly increase it. Thus, with the evidence that elongation inhibitors protect from proteotoxic stress but do not extend lifespan, it is clear that lowering the concentration of newly synthesized proteins is necessary but not sufficient for lifespan extension.C. elegans or to determine the quality of the proteome.The idea that translation inhibitors decrease the speed and improve the coding fidelity is a very interesting suggestion. Unfortunately, we do not have the technical ability to measure the speed of translation in vivo in Reviewer #2 (Recommendations for the authors):C. elegans community is very useful. However, the effect of the compounds used in this study should be clearly shown in C. elegans, as the whole manuscript relies on the comparison of translation inhibition at selected steps. The effectivity of the drugs to inhibit translation initiation and elongation, respectively, was shown using puromycin incorporation , which measures changes in overall protein synthesis and not changes in translation rates at selected steps. The analyzed compounds were studied in other systems such as mammalian cells and yeast, however, the source given in this manuscript does not show if they were previously used and validated in C. elegans. To make the claim more solid that these drugs do inhibit specific steps of translation in the nematode, the authors should have used additional ways to show how the compounds act on the translation process, such as polysome profiling. For example the proposed translation initiation inhibitor salubrinal is suggested to inhibit eIF2alpha phosphatases (hence inducing ISR and reducing translation initiation). However, to my best knowledge, its mechanism of action is unknown and, more importantly, the eIF2alpha phosphatases of C. elegans are not identified, making it speculative if salubrinal does inhibit initiation. Also, while both anisomycin and lycorine are supposed to inhibit elongation and were shown to inhibit overall translation to similar extends , the strength of their effect on survival during heat shock is different between the drugs , raising the question if they indeed act similarly on translation or if they might have off-target effects. For example, anisomycin is discussed to have strong side effects (DOI: 10.1128/MCB.15.9.4930).(1) Validation of the drugs. Using different compounds to inhibit translation at different states is an interesting approach and having these compounds validated for the C. elegans. The reviewers' point that we did not validate them far enough was on point. Addressing this point proved far more difficult and was why revising this paper took so long. We started by synthesizing photoaffinity probes that would have allowed us to pull down and identify the C. elegans targets of these compounds. However, the chemically modified versions of the inhibitors lost their activity, with only the anisomycin probe still behaving like the original molecule.These points are all very well taken. One of the aims of this study was to start validating chemical tools to study translation in Thus we switched strategies and generated the myo-3p::GFP-IRES-tdTomato transgenic worm that allows us to distinguish initiation-dependent from initiation-independent translation. However, as shown in Figure 1F, the variability in fluorescence is large, which caused us difficulties in quantifying the effects precisely. A COPAs bio sorter then allowed us to assay enough animals to unequivocally determine that these inhibitors inhibit translation initiation or elongation as they do in mammals. For 4ER1Cat, we could also show that it no longer extends lifespan in the ifg-1 mutants and that both 4ER1Cat and ifg-1 RNAi surprisingly increase the concentration of newly synthesized proteins in hsf-1 mutants. We thank the reviewer for this criticism, as we think addressing it improved the paper considerably.(2) Consistency of compounds and genotypes used throughout manuscript. Comparing the effects of the inhibition of different translation steps during selected external stresses and in different genetic models is a great approach. Unfortunately, because of the inconsistent use of genotype-treatment-combinations, the lack of Wildtype controls in some assays and splitting graphs based for example on treatment types, it is hard to interpret the results in a global way and to truly compare between initiation and elongation inhibition.In some cases, we were not sure what exactly was meant. All the experiments were conducted in parallel, always including N2 +/- treatment control and focusing on the 4 compounds described. We re-arranged some of the figures and hopefully addressed this point. We initially chose to split thermotolerance, proteasome, and survival graphs to make the different responses more obvious. However, these experiments were conducted in parallel. These are clearly noted as Figures 2B, 2C, 4B, 5A 5B.(3) Statement that elongation inhibitors reduce ongoing toxicity by reducing folding load independently of hsf-1. Some contradictions in the effect of elongation inhibition are not addressed. Elongation inhibition is discussed to reduce toxicity by reducing protein folding load. While it is shown that inhibition of elongation reduces protein aggregation upon heat shock  and in a PolyQ model , protein aggregation is not lowered by elongation inhibition in aged worms . It is further claimed that elongation inhibition acts independently of hsf-1 in many of the observed phenotypes, but sometimes Wildtype controls are missing to rule out a partial dependency. Furthermore, there could be other factors than hsf-1 involved that are changed upon elongation inhibition and mediate the phenotypes instead of (or in addition to) the overall reduction of protein and hence the increase in folding capacity. Also the inhibition of initiation might reduce folding load . More direct data to compare protein aggregation upon inhibition of initiation with elongation during stresses are mainly missing. Translation itself is not compared on a molecular level between the different translation modulation modes.There seem to be several points here. First: It is true that elongation inhibitors could involve other factors. However, the protein folding load model is the simplest version of the model on how elongation inhibitors exert their effect. A key question in this study was to test if we can find conditions where it does not apply. Except for age-associated aggregation, that model correctly predicted all the outcomes for elongation but not initiation inhibitors. For why elongation inhibitors may fail to reduce age-associated protein aggregation please see ANSWER 10 for a potential explanation.Second, we are unsure where we were missing a wild-type control. The only place we could find was the missing images and length quantification for hsf-1 and Q35. There was no detectable difference, and we thus left it out. Regarding going down to the molecular level, we now show in Figure 1 that these drugs act as initiation or elongation inhibitors and establish a new and unexpected role for HSF1 in Figure 6.(4) Effects on lifespan. The lifespan assays in Figure 5 are very interesting and underline the previous observations nicely, as they directly compare two initiation and elongation inhibitors in Wildtype and hsp-1 mutant contexts. With regards to elongation inhibition, there might be contradictions to other published data that found inhibition of elongation using cycloheximide to increase lifespan (DOI: 10.1038/srep18722). One more reason why it would be great to have the effect of the elongation inhibitors on translation used here validated.Thank you for this comment. We were very pleased to find that the inhibitor data, especially the results for 4ER1Cat, agreed so well with the previous results shown by Howard et al. for ifg-1. Such a clear agreement is relatively rare, as genetic or pharmacological inhibition do not necessarily replicate each other. In contrast to knockouts or RNAi, small molecules do not remove the protein but only inhibit a site of a protein. Thus, they should be compared to alleles that affect only one aspect of a protein rather than to knockouts. However, in this case, the pharmacological and genetic results align very well, reciprocally strengthening each other. The hsf-1 dependency of initiation inhibition also aligns very well with Rieckher et al., which we now added to our citation list. The reasons why we did not use cycloheximide and why we think it is problematic to use are outlined in ANSWER 3. As outlined in ANSWER 6, we validated the inhibitor action and appreciate that the reviewer raised this point.(5) Given the high focus of the manuscript on the difference between the inhibition of translation initiation and elongation, translation itself could have been further analyzed using the different compounds and during selected stresses (for example polysome profiling or ribo seq). The authors claim that their results are consistent with a model proposed by Rogers et al. (2011), saying that lifespan extension through inhibition of translation initiation acts via translation of selected factors. Could the status of some of these factors be checked upon translation inhibitor treatment to verify this?This is a great suggestion and we will attempt those for at least two of the inhibitors (anisomycin and 4ER1Cat). However, after the reviewer pointed out that a better characterization of the inhibitors, their mode of action, and targets would be beneficial, we first focused on this action and established that 4ER1Cat acts by inhibiting ifg-1.(6) Figure 2D,E; Figure 4C; Figure 5C,D,E pose multiple questions that remain unanswered: What is the difference between age-associated protein misfolding on the one hand and proteotoxicity caused by heat, proteasome dysfunction, and PolyQ stretches on the other hand, and why do animals react differently to it when treated with initiation or elongation inhibitors? Why does inhibition of initiation protect from age-associated protein toxicity but not from proteasome-misfunction-induced protein toxicity? Why does inhibition of elongation protect from proteasome-misfunction-induced protein toxicity but not from age-associated protein aggregation?These are excellent questions. At the moment, we only have the observation that this seems to be the case. For example, we were surprised that the elongation inhibitors failed to dampen age-associated protein aggregation but could rescue most other forms or proteotoxic stress. We only have a speculative but probably reasonable answer that is testable. As worms age, their translation rates decline. As a result of diminishing translation, the elongation inhibitor has less of an effect with increasing age. Initiation inhibitors, through selective translation, do not only reduce translation but remodel the entire proteome, most likely leading to a less misfolding-prone proteome. While these ideas are currently speculative, we now have chemical tools to time-dependently inhibit translation to test these ideas. For example, if translation declines with age, the protective effects of elongation inhibitors on any of the proteotoxic stressors we investigated in this study should decline as well. In other words, the protective effect of an elongation inhibitor should be proportional to overall translation and thus decline with age.Reviewer #3 (Recommendations for the authors):hsf-1's role in EIs but not IIs protecting against proteasomal dysfunction: the data is Figure 3 make this a weak conclusion at best. In the paradigm used, the difference between WT and hsf-1 mutant survival is only 20% and the 'limited protection' vs. 'highly protective effects' of the respective chemical classes are not apparent. From the figure, the impact seems to be similar on both genotypes. The 'morphology' improvements brought about by EIs are not explained and the images in 3G cannot be used to measure 'unc' phenotypes.There are several aspects to this paragraph. (1) The reviewer is correct in the assessment that the EI improves survival under bortezomib only for hsf-1 and not for N2. Therefore, we have changed the language and now state that the EI provides limited protection in N2.However, we respectfully disagree concerning hsf-1 and polQ. The effects of the EI on the morphology of bortezomib hsf-1 and polQ animals are striking, especially considering that a translation inhibitor would be expected to make animals smaller. The rescue of survival for hsf-1 is also obvious when doing the experiment and reaches p<0.0001. When measured against the DMSO control of the same genotype in hsf-1(sy441) the elongation inhibitors restore survival almost to the level of the hsf-1(sy441) no-bortezomib control .In Figures 2B, C, the data for concluding hsf-1 dependence/independence of IIs and EIs thermotolerance is weak. The graphs look moderately different from each other but only one P value is provided (does it apply to both treatments? what is the comparison?) that is not very convincing (P 0.05 vs. 0.02). No details for means/SEM/number of animals and trials are provided either.To clarify this, we have amended Figure 2 and included animal numbers and P-values.The 0.02 P-value that the reviewer mentions was inserted to show that 4ER1Cat caused a significant increase in survival only at this one-time point in the heat shock assay in the hsf-1(sy441) mutant. While we felt it correct to state that 4ER1Cat is mainly independent of hsf-1 because all other comparisons were not significant, we did not want to ignore this single point. Howard et al. (2016) suggested that the thermotolerance of the ifg-1 mutant (the target of 4ER1Cat) mainly depends on hsf-1 but not wholly. We intended to highlight that our results with a chemical inhibitor match their results with the mutant. In hindsight, this was confusing and made our data look much weaker than they are. In the new version of the figure, we added asterisks to show that the differences are highly significant or not at all. We also specifically state the number of independent experiments included in the figure legends.Data for lifespan  and thermotolerance  survival assays are from one trial only and do not include details . Additional independent trials are critical for verification.This was our mistake. In the revised version, we now clarify these points. For the lifespan data , we provide an extensive supplementary file containing all the lifespan results with ID numbers referenced in the figure to identify the data set in the table that was used to make the figure. All experiments were repeated between 3 to 7 times. We did not sufficiently state that the graph represents the mean of three independent trials. We added the error bars and the total number of animals (sum of three experiments) indicated in Figure 2 for the heat shock data. We also specifically state the number of independent experiments included in the figure legends.”“Data show the mean ± SEM from three independent trials where each measurement is at least: ** = p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001 by row–matched two–way ANOVA with Šídák multiple comparisons test.How were the protein quantifications performed for the Puromycin immunohistochemistry (1A) and the insoluble/soluble fractions . Were specific regions or bands' intensity measured or the whole lane?We have added the following description to the material and methods to address this concern:Line 488:“Gels were quantified in ImageJ by dividing the integrated intensity of each full insoluble lane by the integrated intensity of the corresponding full total protein lane after subtracting a similar area background lane, then normalizing to the DMSO control.”Line 546:“To determine the relative intensities of each blot, the integrated intensity (pixel intensity divided by pixel area) was measured for each lane using ImageJ. A similar-sized band with no signal was used to subtract the background then each intensity normalized to its corresponding GAPDH loading control. Finally, each band’s integrated intensity was normalized to the wild-type DMSO control for quantification and statistics. Significance was determined by one–way ANOVA with Šídák multiple comparisons test.”Is the inhibitor treatment for 72h (3 days) or 4 days? The article mentions 72h  but figures  show 4 days.The reviewer is correct. The exposure is from day 1 to day 4, which is 72h or 3 days. We have amended Figure 2. Figure 1 was substantially changed a no longer contains the day 4 reference.