RNA supply drives physiological granule assembly in neurons

Membraneless cytoplasmic condensates of mRNAs and proteins, known as RNA granules, play pivotal roles in the regulation of mRNA fate. Their maintenance fine-tunes time and location of protein expression, affecting many cellular processes, which require complex protein distribution. Here, we report that RNA granules—monitored by DEAD-Box helicase 6 (DDX6)—disassemble during neuronal maturation both in cell culture and in vivo. This process requires neuronal function, as synaptic inhibition results in reversible granule assembly. Importantly, granule assembly is dependent on the RNA-binding protein Staufen2, known for its role in RNA localization. Altering the levels of free cytoplasmic mRNA reveals that RNA availability facilitates DDX6 granule formation. Specifically depleting RNA from DDX6 granules confirms RNA as an important driver of granule formation. Moreover, RNA is required for DDX6 granule assembly upon synaptic inhibition. Together, this data demonstrates how RNA supply favors RNA granule assembly, which not only impacts subcellular RNA localization but also translation-dependent synaptic plasticity, learning, and memory.

E ukaryotic cells constantly adapt their gene expression in response to specific cellular demands. This happens primarily at the transcriptional level, however, a number of additional mechanisms allow for subsequent post-transcriptional regulation. Post-transcriptional gene regulation crucially contributes to many important cellular processes, e.g. development, differentiation, and maturation of cells in tissues, but also to many pathological alterations. Prominent examples are cancer and neurodegeneration among many others 1-3 . The nervous system sticks out as it displays by far the highest transcriptomic diversity 4,5 . Hence, to enable spatiotemporally controlled protein expression, RNA-binding proteins (RBPs) play crucial roles by guiding mRNAs through the different stages of their life cycle in the cell 6 . RBPs assemble with their target mRNAs into RNA granules or ribonucleoprotein particles (RNPs) 7 . Since their original discovery 8,9 , many different types of RNA granules have been identified and described, e.g. processing bodies (P-bodies), stress granules, and transport granules among others [10][11][12] . Importantly, the content of these RNPs is dynamically regulated by both the demand and supply of RNAs [13][14][15] . While the demand crucially depends on translational activity, the supply of RNAs is additionally determined by transcription, splicing, translational repression, and eventually decay. Consequently, RNPs are able to dynamically assemble and disassemble in order to react to changing cellular demands. This behavior has been nicely visualized by time-lapse fluorescence microscopy 16,17 . In addition to their assembly dynamics, RNPs are very heterogenous in protein and RNA composition 7,18 . Therefore, it is tempting to postulate that a network of RBPs and eventually RNA granules serves as a dynamic buffer system to allow homeostatic RNA metabolism inside the cell 6 . This RNA-based mechanism is a prerequisite for cells to respond to different stimuli in order to remodel the neuronal proteome. Thereby, RNPs balance the RNA demand and supply that is needed for adequate protein synthesis, which ultimately enables cellular plasticity. Disrupting the balance of RNA homeostasis can have important consequences, such as the development of neurodegenerative diseases, which in some cases are hallmarked by pathological deposits of RNA granule components 19 . Here, the concept of liquid-liquid phase separation has an important impact on our understanding of how RNA and RBPs form RNA condensates inside cells 20 . Interestingly, RNA recruitment affects the size and composition of RNAprotein condensates in cells 21 . While the relevance of RNA availability for RNA granule assembly 22 has already been studied in vitro and under diseased or stress conditions, its physiological impact is much less understood. Therefore, we aimed to investigate the assembly and disassembly of RNA granules under physiological conditions in cells. We chose the RNA helicase DDX6 (Rck, p54), which has been shown to mediate translational suppression of specific mRNAs in P-bodies 23,24 . Notably, DDX6 is not only found in P-bodies, but also in other types of RNA granules, including stress granules and Staufen2 (Stau2) containing transport RNPs 18 . The yeast homolog Dhh1 regulates RNP assembly and turnover 25 . Together, this underlines the importance of DDX6 in gene expression regulation 18,26 . Here, we report that DDX6 granule formation is regulated by RNA supply in the neuronal cytosol, as well as neuronal maturation and synaptic activity.

Results
Cytoplasmic DDX6 granules physiologically disassemble during neuronal maturation. To get first insight into the regulation of DDX6 granule formation, we took advantage of primary neurons in cell culture as a model system. As hippocampal neurons undergo immense morphological and physiological changes during their maturation 27 , we speculated that these alterations might influence RNA granule properties as well, and studied DDX6 granule formation at different stages of maturation 28 . We observed cells with larger and smaller DDX6 granules at all observed time points during maturation, though at different proportions. To quantify this variability in the cell population, we manually counted > 100 cells per time point and biological replicate by eye and categorized cells into two groups: cells containing either preferentially large or small granules. Examples of this quantification are provided in Suppl. Fig. 1a. We found a striking shift in the population from immature hippocampal neurons with large granules to mature neurons with small granules, indicating significant reorganization (Fig. 1a, b; F 3,8 = 0.0044). This occurred in a similar fashion during the maturation of cultured cortical neurons (Suppl. Fig. 1b). To determine whether this effect was specific to DDX6 granules or rather reflected global remodeling during neuronal maturation, we investigated granule formation of 6 other RBPs with diverse functions (i.e. ZBP1, UPF1, RBM14, Pur α, Pum2, Mov10) (Suppl. Fig. 1c). Interestingly, we observed various distinct localization patterns. However, none of these RBPs displayed a change in granule formation comparable to that of DDX6, indicating an RBP specific effect. Detailed single particle quantification of DDX6 revealed a decrease in granule size (p = 0.0452; 8 days in vitro (DIV) vs. 29 DIV), but an increase in their number (p = 0.0081), further corroborating RNA granule reorganization (Fig. 1c, d). Mature neurons showed a smaller variability in granule size within single cells compared to immature neurons, suggesting that granules were not only smaller but also more homogeneous (Suppl. Fig. 1d). Additionally, we observed a shift of DDX6 immunofluorescence from granules to the cytosol compartment during maturation ( Fig. 1e; p = 0.00096). The observed disassembly was accompanied by decreased DDX6 protein levels, both in hippocampal (Suppl. Fig. 1e) and cortical neurons (Suppl. Fig. 1f). To confirm that these observations hold true in the living animal, we performed DDX6 immunostainings on brain slices of 8 day old and 10 month old mice. Indeed, we detected a clear reduction in DDX6 granule size in mature compared to young mice, both in the hippocampus and cortex (Fig. 1f, Suppl. Fig. 1g). To gain insight into granule dynamics in living cells, we performed live imaging of green fluorescent protein (GFP) fused to DDX6 (GFP-DDX6) transiently expressed in hippocampal neurons. Granules generally displayed diffusion-like mobility and fused or split in individual instances, providing first insight into how granule assembly can be regulated over a longer time period (Suppl. Fig. 1h and Suppl. Movie 1). This is in line with a recent publication, showing that the assembly of cytoplasmic condensates is regulated by coalescence in vivo 29 . Taken together, our findings clearly show that physiological DDX6 granules are remodeled during neuronal maturation in vivo.
Neuronal activity regulates DDX6 granule assembly in mature hippocampal neurons. Next, we asked the question how synaptic activity, a key physiological hallmark of neuronal maturation 28 , might alter the properties of DDX6 granules, as previously suggested for other types of RNPs 13,17 . Here, we interfered with synaptic activity by simultaneous inhibition of AMPA receptors, NMDA receptors, and voltage-gated sodium channels through combined application of CNQX, AP5, and TTX 30 (subsequently termed silenced) and investigated its impact on DDX6 granule formation. Interestingly, the inhibition of synaptic activity in mature neurons resulted in the reassembly of DDX6 into larger granules (Fig. 2a, b; p = 0.0012). Detailed single particle analysis revealed a significant increase in average granule size and a decrease in the number of granules in the cell body ( Fig. 2c; Fig. 2d; p = 0.00010). Importantly, these newly formed assemblies were not stress granules, nor did this treatment induce stress granules (Suppl. Fig. 2a). Reinstating endogenous neuronal activity by wash-off of inhibitors ('recovery') resulted in the quick reversal of granule assembly within fifteen minutes ( Fig. 2e; F 2,18 = 1.59e-05, treatment 1; F 2,18 = 2.38e-11, treatment 2). Additionally, DDX6 disassembly could be rapidly induced by synaptic activation of NMDA receptors, independent of the prior treatment. To gain further insight into how granules are disassembled upon NMDA treatment, we transiently expressed GFP-DDX6 in hippocampal neurons and performed live imaging during NMDA application and subsequent NMDA wash-off (Suppl. Fig. 2b and Suppl. Movie 2). We observed DDX6 granules gradually disassemble during NMDA treatment and conversely reassemble upon wash-off, a process distinct to the fusion and splitting of granules observed at baseline neuronal  Fig. 1h and Suppl. Movie 1). Together, these experiments revealed that DDX6 granule assembly is dynamically regulated by synaptic activation or inhibition.
Stau2 promotes DDX6 granule assembly upon neuronal inhibition. Synaptic activity crucially determines the localization and translatability of mRNAs 31,32 . Therefore, we speculated that synaptic transmission might modulate DDX6 granule formation by the availability of transcripts. To test whether RBPs involved in RNA transport would supply RNAs needed for DDX6 granule formation, we focused on the double-stranded RBP Stau2, which we have previously identified as an interaction partner of DDX6 in neurons 18 . Stau2 recognizes complex RNA secondary structures predominantly in the 3' untranslated region (UTR) of its targets and is an essential RNA transport protein responsible for localizing a significant fraction of the neuronal transcriptome within different cellular compartments 30,[33][34][35][36] . A DEAD box RNA helicase such as DDX6 may play a role in the regulation of such complex targets. Moreover, Stau2 can transiently interact with other types of RNA granules in living neurons 17 suggesting that it serves as a dynamic RNA distributor and regulator in cells. To investigate whether Stau2 indeed associates with DDX6 in the cell body of neurons, we performed co-immunostainings (Fig. 3a). Though Stau2 and DDX6 generally displayed distinct localization patterns, we observed instances where these two proteins clearly colocalized, indicating transient interactions may occur. To test for its functional contribution to the assembly of DDX6 granules in mature neurons, we depleted Stau2 protein from cells using lentiviral transduction of a short-hairpin RNA targeting Stau2 mRNA 34 (Suppl. Fig. 3a, b). This reduction in   Fig. 3c) upon neuronal inhibition was prevented in Stau2 depleted cells. Together, this data indicates that Stau2 is required for dynamic DDX6 granule assembly upon neuronal inhibition, raising the possibility that the regulation of mRNAs is a determining factor in this process.  DDX6 granule assembly is facilitated by the supply of cytoplasmic non-translating mRNAs. Next, we inquired if translational activity, which determines whether an mRNA is covered by ribosomes or accessible for RNA granule formation, affects RNA supply to granules. Translational inhibitors have been shown to affect RNA granule size [37][38][39][40] . To test whether RNA availability is indeed crucial for DDX6 assembly in neurons, we exploited three different translation inhibitors: puromycin (PMY), harringtonine (HRN), and cycloheximide (CHX). Though all three compounds inhibit translation, they differ in their underlying mechanism, as PMY dissociates translating ribosomes and releases the bound transcript, HRN blocks initiating ribosomes and thereby induces ribosome runoff, whereas CHX results in stalling and stabilization of ribosomes on mRNAs [41][42][43] . Thus, all three inhibitors have a different impact on the pool of mRNAs that are available for granule formation; while PMY and, in a translation speed dependent manner, HRN eventually increase the pool, making mRNAs abundant in the cytoplasm and available for other interactors, CHX restricts the number of assembly competent RNAs. We found that PMY treatment resulted in fast DDX6 granule assembly (Fig. 4a, b, p = 0.02769). Additionally, HRN treatment caused a gradual assembly upon prolonged incubation (Fig. 4d, e; F 3,8 = 0.00333), possibly due to the induction of ribosome runoff that depends on translational speed (Suppl. Fig. 4a). Detailed single particle analysis of PMY and HRN treated neurons revealed a significant increase in average DDX6 granule size ( Fig. 4c; p = 0.0022, Fig. 4f; p = 0.0096) and decrease in granule number (Suppl. Fig. 4b; p = 0.0145, Suppl. Fig. 4c; p = 0.0273) in both cases. Conversely, neurons treated with low concentrations of CHX showed a moderate decrease in the neuronal population containing large DDX6 granules (Suppl. Fig. 4d, e). This small effect is likely due to the fact that granules are generally already quite small at baseline in 22 DIV neurons. To better assess the effects of CHX at 22 DIV and to inquire whether it acts upstream or downstream of neuronal activity, we first inhibited neuronal activity to induce large granules and subsequently applied CHX as shown in Fig. 4g. Interestingly, large DDX6 granules induced by neuronal inhibition disassembled when cells were treated with low concentrations of CHX (Fig. 4g, h; p = 0.00084), opposing the assembly promoting effect of neuronal inhibition. Again, these assemblies did not represent stress granules, nor did the treatments induce stress granules on their own (Suppl. Fig. 4f). As DDX6 granules assemble upon the supply of cytoplasmic non-translating mRNA, we were curious to know whether DDX6, a well-studied RNA helicase implicated in translation 44 , might interact with translating mRNAs or ribosomes. Therefore, we performed polysome profiling in developing and mature cortical neurons (Suppl. Fig. 4g). DDX6 was clearly depleted from polysome fractions in both conditions, indicating that the protein either interacts mainly with non-translating mRNA or only transiently interacts with polysomes. Together, our data demonstrate that balancing RNA availability and translation activity crucially regulate DDX6 granule formation in neurons.
Depletion of RNA from DDX6 granules reduces their assembly capability. Having established that RNA availability is essential to promote DDX6 granule assembly in living cells, we investigated whether RNA supply is indeed needed for dynamic DDX6 granule assembly/disassembly. To further investigate the effects of RNA depletion specifically in DDX6 granules in the living cell, we generated a reporter construct consisting of GFP fused to DDX6 (GFP-DDX6), which was C-terminally tagged with the endonuclease RNase1 (Fig. 5a), to effectively deplete RNA from DDX6 granules. We first confirmed that DDX6 granules were accessible for RNase1 digestion, by exploiting differential centrifugation combined with an in vitro RNase1 treatment (Suppl. Fig. 5a). To verify that the tagged RNase1 was indeed enzymatically active, GFP-DDX6 and GFP-DDX6-RNase1 were expressed in HEK cells, the expressed fusion protein enriched by anti-GFP immunoprecipitation, and incubated with purified RNA. Here, we observed a clear reduction of intact RNA in samples incubated with GFP-DDX6-RNase1 compared to GFP-DDX6 (Suppl. Fig. 5b, c). Next, we transiently transfected GFP-DDX6 or GFP-DDX6-RNase1 reporters into hippocampal neurons (Fig. 5b). Both reporters formed granules that contained DDX6 and most often DCP1a, a protein commonly associated with DDX6 45,46 , as shown by immunostaining. Neurons containing the GFP-DDX6-RNase1 reporter showed a small but clear reduction in GFP granule size (Fig. 5c, p = 0.0306; Suppl. Fig. 5d) and a nonsignificant increase in granule number (Fig. 5d), compared to GFP-DDX6 transfected cells. Importantly, these effects resembled the rearrangement of endogenous DDX6 granules observed in neurons during maturation (Fig. 1). Notably, both GFP-DDX6 and GFP-DDX6-RNase1 reporters were expressed at similar levels (Suppl. Fig. 5e). To confirm, that this effect is in fact due to RNA depletion in granules, rather than global degradation, we generated two additional RNase1 reporters that do not localize to DDX6 granules: (i) RNase1 fused to RFP (RFP-RNase1) freely diffusing in the cytoplasm, and (ii) RNase1 fused to RFP N-terminally tagged with the first 99 nucleotides of the outer mitochondrial membrane protein TOMM20 (TOMM20-RFP-RNase1), resulting in the tethering of the reporter to the outside of mitochondria (Suppl. Fig. 5f) 47,48 . Equivalent reporters lacking the RNase1 tag were used as control. Both TOMM20-RFP reporters co-localized strongly with the mitochondrial protein cytochrome C (CYT C), spatially distinct to DDX6 granules (Suppl. Fig. 5g). The four RFP constructs were co-transfected with the GFP-DDX6 reporter to assess their effect on DDX6 granules (Suppl. Fig. 5h). None of the RFP reporters affected DDX6 granule size, number, or fluorescence intensity (Suppl. Fig. 5i-k). Together, these experiments indicate that RNase1 activity outside DDX6 granules does not account for the reduction in DDX6 granule size we observed by its direct tethering. This strongly suggests that RNA degradation at DDX6 granules drives their disassembly. Finally, we investigated whether the changes in DDX6 granule assembly dependent on neuronal activity (Fig. 2) would also require RNA supply. To this end, we transfected either our GFP-DDX6 or GFP-DDX6-RNase1 reporters in mature hippocampal neurons (Suppl. Fig. 5l) and inhibited neuronal activity overnight by combined application of CNQX, AP5, and TTX. We observed a clear increase in GFP-DDX6 granule size upon neuronal inhibition, validating our previous findings (Fig. 2) and confirming our overexpressed reporter phenocopied endogenous DDX6 ( Fig. 5e; F 1,8 = 0.00045, GFP reporter; F 1,8 = 0.0128, neuronal inhibition). Interestingly, the smaller granules formed by GFP-DDX6-RNase1 did not significantly increase in size after neuronal inhibition (Fig. 5e). This strongly supports the notion that the assembly of DDX6 granules governed by neuronal inhibition requires RNA supply. Notably, the average number of GFP-DDX6 and GFP-DDX6-RNase1 granules remained largely unchanged in the cell body (Suppl. Fig. 5m).
To gain initial insight into whether DDX6 is involved in the regulation of synaptic function, we performed two pilot experiments with hippocampal neurons lacking DDX6. This assumption is based on the fact that DDX6 has been linked to translational regulation, an essential process for synapse formation 49,50 . Hippocampal neurons were transduced using a lentivirus expressing a short-hairpin RNA targeting DDX6 mRNA (shDDX6) resulting in a~40% reduction of DDX6 protein and DDX6 granule size, mimicking the reduction in protein levels and granule size observed during neuronal maturation (Fig. 1a-c and Suppl. Fig. 1e, f). Firstly, downregulation of DDX6 led to increased clustering of the postsynaptic marker Homer1 in dendrites of 15 DIV hippocampal neurons (Suppl. Fig. 5n, o). Secondly, Ca 2+ live imaging showed an increase in the frequency of fluorescent peaks in neurons where DDX6 was depleted (Suppl. Fig. 5p, q; p = 0.043, Suppl. Movie 3). Together, these preliminary data indicate that DDX6 is likely involved in the regulation of synaptic function. We propose that depletion of DDX6 may de-repress synaptically relevant mRNAs, resulting in de-regulated synapse formation and activity. Future In summary, our results clearly underpin the importance of RNA for granule assembly in cells. Moreover, we show that the process of DDX6 complex formation is highly dynamic and relies on a fine-tuned balance between RNA supply and translational activity that is modified by synaptic activity and neuronal maturation.

Discussion
Our work provides functional and mechanistic insights into how RNA granules might dynamically balance transcriptomic and translational changes and could respond to the demand for the translation of specific transcripts. Mammalian cells are able to shape protein expression by regulating the translatability of their mRNAs 51,52 . In this respect, several independent mechanisms such as RNA transport 53 , binding of translation regulators 6 , or RNA degradation 54,55 have evolved to allow spatiotemporal sorting of mRNAs and the control of protein expression patterns. Recent work has shown that mRNAs facilitate condensation into RNA granules in order to regulate their expression in vitro 22,56,57 . This goes well in line with our finding that DDX6 granule assembly is affected by processes regulating the amount and availability of cytosolic RNA, such as translation activity and synaptic transmission. This mechanism involving the DEAD box RNA helicase DDX6 seems to be evolutionarily conserved, as a parallel study in Drosophila 58 found that granule formation of the DDX6 homolog Me31B during aging affects the translatability of mRNAs in the fly brain. Together, this indicates that DDX6 granules regulate transcripts during neuronal maturation and synaptic transmission by the assembly and disassembly of RNA granules. Though DDX6 and P-bodies have been extensively studied as translational repressors and regulators of mRNA and DDX6 is a core component of P-bodies 11,46 , it remains to be seen whether DDX6 function itself directly affects RNA-dependent granule assembly and disassembly. Moreover, we do not yet know whether DDX6 binds free mRNA in a non-specific manner, or rather interacts with specific targets during the assembly processes observed in our study. Future studies will have to unravel how this DEAD box RNA helicase conveys RNP assembly in neurons and how this process is regulated by maturation and synaptic activity. Indeed, DEAD-box ATPases have been identified as regulators of RNA granule formation 25,59 . Interestingly, eIF4A, another RNA helicase in mammals, has been implicated in the regulation of RNA-RNA interaction dependent assembly, and limits the formation of RNA condensates 15 . These findings are in line with an in vitro study showing that RNA can condensate through intermolecular RNA-RNA interactions 56 .
In addition to RNA availability, we have recently identified RNA secondary structures as another important feature in the process of RNA granule assembly. Here, specific RNA hairpins bound by Stau2 regulate granule assembly, translatability, and dendritic transport of target RNAs 36 . Consequently, RNAs can drive granule assembly not only through sequences but also through secondary structures in vivo. Here, we show that the condensation of DDX6 granules requires Stau2 dependent RNA regulation during synaptic inhibition. This is most likely due to the involvement of Stau2 in the transport and redistribution of mRNAs. However, we cannot exclude that the translational regulation of specific target mRNAs by Stau2 may contribute to this process as well. Together, our data clearly support a working model in which RNA supply and neuronal activity critically determine DDX6 granule formation in cells. In turn, depletion of DDX6 results in altered synaptic activity. This mechanism of RNA granule assembly would allow cells to cope with different physiological conditions and to dynamically balance the demand for the translation of specific transcripts, via the reversible assembly of RNA granules. Therefore, the study of RNA granule formation is important for our understanding of physiological processes, such as synaptic plasticity in neurons, which heavily depends on mRNA regulation 60,61 . Moreover, unraveling the underlying molecular principles of RNA granule assembly is a prerequisite for our understanding of the physiological impact of foreign RNA exposed in a cell, e.g. complex RNA viruses such as SARS-CoV-2 or the efficiency of mRNA vaccines 62,63 .

Methods
Neuronal cell culture and transfection. Primary rat hippocampal and cortical neuronal cell cultures were generated as previously described 64 . In short, hippocampi of embryonic day 17 (E17) embryos of timed pregnant Sprague-Dawley rats (Charles River Laboratories) were isolated, cells dissociated, and plated on Poly-L-Lysine coated coverslips and cultured in NMEM + B27 medium (Invitrogen). Experiments were performed with cultured neurons between 8-29 days in vitro (DIV). Neurons were transiently transfected by calcium phosphate co-precipitation as previously described 65 . A total amount of 3 µg DNA was used for single or cotransfected plasmids. All animals were used according to the German Welfare for Experimental Animals (LMU-Munich, Regierung von Oberbayern).
Microscopy. Imaging of sagittal brain sections was performed at the core facility bioimaging of the Biomedical Center with an inverted Leica SP8 microscope, equipped with a Plan-Apochromat 63x objective, a laser for 552 nm excitation, hybrid photo detectors (HyDs), and a convectional photomultiplier tube (PTM; Hamamatsu R 9624). Image pixel size was 361 nm. Z-stacks were acquired at 700 nm interval. Imaging of fixed cells was performed on a Zeiss Z1 Axio Observer microscope, including a Plan-Apochromat 63x objective, a COLIBRI.2 LED light source and the Axiocam 506 mono camera. Live cell imaging was performed on a Zeiss Cell Observer spinning disk system, consisting of a Zeiss Z1 Axio Observer microscope including a Plan-Apochromat 63x objective, a Yokogawa CSU-X1 spinning disk unit with 4 laser lines (405 nm 20 mW; 488 nm 50 mW, 561 nm 75 mW and 638 nm 75 mW) and an Evolve 512 Delta EMCCD Camera. A custom made EMBL environmental chamber (EMBLEM) was used for temperature control. Hippocampal neurons were imaged at 36.5°C in HBSS (Life Technologies) supplemented with 20 mM HEPES buffer pH=7.3 (Sigma Aldrich). Time-lapse images were acquired at an approximate frame interval of either 30 or 0.5 sec. Z-stacks at 1 µm intervals spanning the entire cell body were acquired per timepoint for live imaging of NMDA and wash-off treatments, and subsequently subjected to maximum intensity projection. Cells were selected for proper expression of plasmid as well as for cell morphology and cell viability.
Image data analysis. Assessment of neuronal population with large or small granules (categorized as exemplified in Fig. 1a by 8 DIV vs. 29 DIV, and Suppl. Fig. 1a) was done by manually scoring >100 cells/condition/experiment. Detailed quantification of granule size and number was performed using the thresholding function of the Arivis Vision 4D software. Quantification of granular vs. cytoplasmic fluorescence was performed with a custom written ImageJ 70 script, available upon request. In short, images were gamma adjusted, a Laplacian filter was applied and images were thresholded using the automatic MaxEntropy method, to generate a detection mask for the granular compartment. A second detection mask for an approximation of cytoplasmic intensity outside granules was NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-30067-3 ARTICLE NATURE COMMUNICATIONS | (2022) 13:2781 | https://doi.org/10.1038/s41467-022-30067-3 | www.nature.com/naturecommunications generated by dilating the first mask by 2 pixels and subtracting the first mask, leaving only the dilated part outside granules. Both masks were used to measure the mean fluorescence in the original unprocessed images.
For deconvolution, z-stacks were acquired at a distance of 0.26 µm. Z-stacks were subjected to deconvolution using the constrained iterative quantitative restoration method of the Zeiss ZEN software deconvolution module.