Drosophila Clueless ribonucleoprotein particles display novel dynamics that rely on the availability of functional protein and polysome equilibrium

Drosophila ovaries as a model to study Clu bliss particle dynamics

To identify the protein domains and protein synthesis requirements of Clu particle dynamics, we examined particle assembly and disassembly in female germ cells using fixed and live imaging. Clu particles are always present in the nurse cells of well-fed follicles (Cox & Spradling, 2009; Sheard et al., 2020). However, they look larger or smaller, or more or less numerous, depending on imaging conditions, including the length of dissection, whether the tissue is imaged while fixed or live, and the type of microscope (spinning disc vs laser confocal). However, importantly, Clu particles completely disassemble in response to stress (Fig 1A’, B’), thus we used a binary decision for assembly/disassembly – either Clu particles were present or absent – to determine how various conditions affect dynamics. Drosophila females have a pair of ovaries composed of 16-20 ovarioles (Fig 1D, E, (Spradling, 1993) (Sarikaya et al., 2012)). Ovarioles are strings of developing follicles that contain all follicle stages (2-14) when dissected from well-fed females. For consistency, we predominantly analyzed data from Stage 6 & 7 follicles, which are large enough to be readily imaged and contain many Clu particles but are previtellogenic, occurring before many complex developmental events (Fig 1E, S1 Table, see Materials and Methods for additional details regarding imaging).

The DUF, Clu, and TPR domains are necessary for Clu bliss particle association

Clu is a large, multi-domain protein (Fig 1C); however, the function of each domain and how they control particle dynamics is poorly understood. The ms (melanogaster-specific) domain is unique to Drosophila and is not required to rescue the clu null mutant (Sen & Cox, 2016). The ßGF (beta Grasp Fold) domain is so-called based on the predicted structure, and the DUF (Domain of Unknown Function) is predicted based on sequence homology (Sen et al., 2015). The Clu domain is highly conserved, but we do not yet understand its role. We previously demonstrated that the TPR (tetratricopeptide repeat) domain is critical for Clu’s binding mRNA (Sen & Cox, 2016). Finally, the M (Middle) domain sequence is unstructured. While it does not contain traditional intrinsically disordered (ID) motifs, ID motifs are characteristic of proteins associated with membraneless organelles (Alberti et al., 2019; Li et al., 2012; Shin & Brangwynne, 2017).

Previously, we used co-immunoprecipitation to show that full-length Drosophila Clu can self-associate (Sen & Cox, 2016). To test which domains are required for Clu particle assembly in vivo, we used the GAL4/UAS system. We created transgenic lines that ectopically express Clu-tagged with mScarlet at the C-terminus under the control of the conditional UASp promoter (Fig 2A). To simultaneously visualize ectopically expressed mScarlet-labeled constructs and endogenous Clu, each construct was combined with nanos GAL4 (nosGAL4) in a clu⁰⁶⁶⁰⁴ background which expresses Clu::GFP (Fig 1C). The nosGAL4 line we used is clonally expressed in the nurse cells (Hanyu-Nakamura et al., 2004). Using live imaging, we found full-length (FL)-Clu::mScarlet reliably formed Clu particles (Fig 2B’), which co-localized with endogenous Clu::GFP particles, indicating that both Clu species exist within the same particle (Fig 2B”, S1 Movie). To determine if the DUF, Clu, and TPR domains are required for particle assembly, we ectopically overexpressed mScarlet-labeled Clu transgenes with each domain deleted (ΔDUF::mScarlet, ΔClu::mScarlet, and ΔTPR::mScarlet) (Fig 2A, C-E”, S2-4 Movies). Using live imaging, we were unable to see particle assembly of any of these deletion constructs (Fig 2C’, D’, E’). Furthermore, none of these deletions co-labeled with endogenous Clu::GFP particles (Fig 2C”, D”, E”). This suggests that these three domains are necessary to assemble Clu particles and to associate with already assembled endogenous particles.

Bliss particles disassemble in response to high expression of functional Clu

When we tested expression levels of ectopic FL-Clu using different GAL4 drivers, we noticed a dose-dependent effect on particle formation. daughterless (da) GAL4 induced high uniform expression of Clu in germ cells (Fig 3A-A”) (Casper et al., 2011; Morawe et al., 2011). In contrast to nosGAL4, when ectopic FL-Clu::mScarlet was expressed by daGAL4 (Fig 3A’), neither ectopic Clu nor endogenous Clu assembled into particles (Fig 3A vs 2B-B”). To test whether this was dependent on functional FL-Clu, we ectopically overexpressed Scarlet-labeled ΔDUF or ΔTPR domain (ΔDUF::mScarlet, ΔTPR::mScarlet, Fig 2A) using daGAL4. Consistent with our observations using nosGAL4, neither was able to assemble mScarlet particles (Fig 3B’, C’), but unlike ectopic FL-Clu, ΔDUF and ΔTPR overexpression did not disassemble endogenous Clu particles (Fig 3B, C). Finally, to ensure that expressing high concentrations of any protein does not cause cell stress that inhibits particle assembly, we overexpressed unrelated mCherry-labeled Capping Protein Beta (mCherry::CPB) (Ogienko et al., 2013). mCherry::CPB also did not disrupt endogenous Clu particles (Fig 3D-D”). This suggests that the mechanism assembling Clu particles at least partly depends on regulating the concentration of functional Clu but does not respond to non-functional Clu (ΔDUF and ΔTPR).

The translation inhibitor puromycin disassembles Clu bliss particles

Stress granules and P-bodies have distinct responses of assembly/disassembly in response to various stressors and different translation inhibitors, depending on the drugs’ mechanism (Buddika et al., 2020; Eulalio et al., 2007; Patel et al., 2016). This is thought to be due to manipulation of available levels of associated messenger RNPs (mRNPs) and polysome presence and activity (Hofmann et al., 2021; Hubstenberger et al., 2017; Ivanov et al., 2019; Kato & Nakamura, 2012). Puromycin (PUR) is a commonly used and well-studied translation inhibitor. PUR forms a stable peptide bond with nascent polypeptides, resulting in premature translation termination which leads to ribosome complex disassembly, polysome loss and increased free mRNAs (Aviner, 2020) (Fig. 4A). Since Clu is a ribonucleoprotein critical for mitochondrial function that binds mRNAs encoding mitochondrial proteins, we wanted to determine how puromycin treatment affected bliss particle dynamics. To do this, we treated ovarioles dissected from well-fed clu⁰⁶⁶⁰⁴ females with PUR and found that the plentiful bliss particles were quickly and completely disassembled within ten minutes (Fig 4B-C’, S5 Movie). Given the molecular action of PUR, this suggests that disassembled ribosomes and/or increased concentrations of mRNPs cause Clu particles to disassemble.

Cycloheximide treatment maintains Clu bliss particles, but blocks insulin-induced assembly

CHX is another well-known and frequently used translation inhibitor that binds to the exit site of the ribosome which stalls the ribosome and blocks translation elongation, leading to increased concentrations of polysomes (Fig 5A) (Duncan & Mata, 2017). Under normal conditions, CHX treatment causes P-bodies to disassemble (Eulalio et al., 2007; Kedersha et al., 2000; Lin et al., 2008; Moutaoufik et al., 2014; Patel et al., 2016). To determine the effect of CHX on bliss particles ex vivo, we tested whether CHX treatment disassembled particles and found that this was not the case (Fig. 5B-C’). To ensure our method of CHX treatment was effective, we dissected ovarioles from well- fed trailer hitch^CA06517 (tral) females that express GFP inserted at the endogenous tral locus, thus labeling P-bodies (Buszczak et al., 2007; Eulalio et al., 2007; Kato & Nakamura, 2012). We confirmed that Tral-labeled P-bodies decreased in size and number with CHX addition compared to mock control, as previously shown (S1 Fig) (Eulalio et al., 2007; Patel et al., 2016).

Ex vivo imaging has many advantages for investigating cellular dynamics. However, we wanted to determine the effect of CHX on Clu bliss particles in vivo. To do this, we switched well-fed females to yeast paste supplemented with CHX for 24 hours. Feeding CHX has been shown to reduce protein synthesis (Hwang & Cox, 2024). To determine the effect of dietary CHX on Clu bliss particle dynamics in vivo, we fixed and immunolabeled ovarioles from CHX-fed females (Fig 5D-F”). As expected, untreated well-fed females displayed robust Clu particles (Fig 5D, D”). Like our ex vivo experiment, CHX-fed females also exhibited robust Clu particles, indicating that CHX feeding does not disassemble particles (Fig 5E, E”, F, F”). We previously demonstrated that mitochondria mislocalize in nurse cells when the females are exposed to various stressors (Cox & Spradling, 2009; Sen & Cox, 2016; Sen et al., 2015; Sheard et al., 2020). CHX-feeding not only maintained bliss particles, but also maintained mitochondrial morphology and distribution, supporting that feeding CHX was not stressful for the nurse cells (Fig 5E-F’’).

Since CHX did not disassemble bliss particles ex vivo and in vivo, we wanted to test the effect of CHX on particle assembly. To do this, we dissected ovaries from starved clu^CA06604 females that have completely disassembled bliss particles (Fig 5H, I, S8-9 Movie) (Sheard et al., 2020). Normally, adding insulin to the media causes bliss particles to quickly assemble (Fig 5H, H’, S8 Movie, Sheard 2020). However, with preincubation of CHX, insulin-induced bliss particle assembly was blocked (Fig 5G, I, I’, S9 Movie). Taken together, these data suggest that the stalled ribosomes and increased polysomes occurring with CHX treatment do not disassemble already formed bliss particles. However, insulin signaling is insufficient to drive particle assembly when ribosomes are stalled in the absence of particles.

Cycloheximide maintains bliss particles in the presence of nutritional stress

For effective Drosophila ex vivo imaging, insulin must be added to the media to fully support the tissue (Morris & Spradling, 2011). If it is omitted, egg chamber development is not normal, and the samples start to degenerate (Prasad et al., 2007; Prasad & Montell, 2007). Since Drosophila Insulin-like peptides secreted by the brain are required for normal egg chamber development, incubating follicles without insulin does not supply effective nutritional signaling and is stressful to the tissue (LaFever & Drummond-Barbosa, 2005; Richard et al., 2005). Since CHX did not abolish bliss particles, in contrast to PUR, we wondered whether CHX treatment could confer a protective effect to maintain bliss particles in the presence of stress. Ovarioles, which were dissected from well-fed clu^CA06604 females and pre-incubated in insulin-free media (CS), disassembled bliss particles within 30 minutes, supporting the critical role of nutrition in maintaining particles (Fig 6B, B’). Surprisingly, adding CHX ex vivo after dissection was sufficient to maintain bliss particles for 30 minutes, even in the absence of insulin (Fig 6B”). To test whether CHX could also protect bliss particles from nutritional stress in vivo, we starved flies after feeding CHX. Well-fed w¹¹¹⁸ females were switched to wet yeast paste containing CHX and fed for 24 hours. They were then starved on water only for 5 hours. Five hour starvation completely disassembled bliss particles (Fig 6C, C”) (Sheard et al., 2020). However, providing CHX for 24 hours before starvation was sufficient to maintain bliss particles and protect them from disassembly (Fig 6D, D”, E, E”). Not only are particles maintained, CHX feeding before starvation appears to decrease cellular nutritional stress as indicated by normal mitochondrial localization (Fig 6C’ vs D’, E’) (Sheard et al., 2020). This observation supports that stalled ribosomes and increased polysomes maintain and protect bliss particles even with decreased nutrition ex vivo and in vivo. Decreased protein synthesis also appears to protect the nurse cells from nutritional stress-induced mitochondrial mislocalization.

Cycloheximide maintains bliss particles in the presence of oxidative stress

Hydrogen peroxide (H₂O₂) is highly toxic to cells, immediately causing a sharp increase in reactive oxygen species and oxidative damage (Vona et al., 2021). Previously, we demonstrated that adding H₂O₂ to cultured ovarioles quickly disassembles Clu bliss particles (Fig 7B-B”’, S10 Movie) (Sheard et al., 2020). Since CHX treatment of cultured ovarioles maintained Clu bliss particles even in the absence of insulin (Fig 6B”), we tested if CHX treatment could maintain bliss particles with high levels of oxidative stress (Fig 7). We cultured ovaries from well-fed clu^CA06604 females, added CHX, and then exposed the ovarioles to H₂O₂ (Fig 7A). Surprisingly, adding CHX maintained Clu bliss particles in the presence of H₂O₂ (Fig 7C-C”’, S11 Movie). This was also observed for a higher CHX concentration (Fig 7D-D”’, S12 Movie). This observation suggests that CHX treatment and increased polysomes are sufficient to maintain and protect Clu bliss particles from oxidative stress-induced particle disassembly, in addition to protection from nutritional stress.

Clu particles associated with mitochondria move more slowly

Because we observed that CHX treatment had an impact on bliss particle assembly and disassembly, we tested whether CHX treatment affects bliss particle velocity (Fig 8, S6-7 Movies). Using kymographic analysis, we tracked the movement of the directed Clu particles and measured their velocities (Fig 8B, B’, white arrows). The average particle velocities were not affected by CHX treatment (Fig 8C), indicating that modulating translation activity by CHX did not impede particle movement although CHX affected bliss particle assembly/disassembly. Previously, we demonstrated that Clu particles are juxtaposed to, but not overlapping with, mitochondria in fixed tissues (Cox & Spradling, 2009). However, we did not investigate whether this mitochondrial association is related to particle movement. To answer this question, we live-imaged follicles dissected from clu^CA06604 females and treated them with the mitochondrial membrane potential sensitive dye tetramethylrhodamine ethyl ester (TMRE) to visualize mitochondria. Clu particles frequently moved in the cytoplasm while associated with mitochondria (Fig 9A-E, S13 Movie). Using kymographic analysis, we tracked the speed of mitochondrial-associated and unassociated Clu particles (Fig 9B, C, E) and measured particle size (Fig 9A, D, E). The Clu particles associated with mitochondria moved significantly slower than the particles not associated (Fig 9C). Particle size did not correlate with whether or not the particle was associated with a mitochondrion (Fig 9D). However, as particle size increased, the speed decreased (Pearson correlation, r = −0.3287, Fig 9E). To confirm that particle size did not affect mitochondrial association, we performed logistic regression analysis (Fig 9F, G, S2B, C). Using simple logistic regression analysis (one independent variable: size or speed), we found that the speed of particle movement greatly affected mitochondrial association, but Clu particle size did not affect whether the particle was mitochondria-associated (Fig 9F vs. G, S2B vs. C). This suggests that the speed of the particle, not the size, determines whether there is particle-mitochondrion association.

Clu bliss particle assembly requires regulated levels of functional Clu

Clu could act as a scaffold protein within particles as this is a common feature for cytoplasmic granule proteins (Buchan, 2014). Many cytoplasmic bodies and granules have associated proteins containing low-complexity or prion-like domains. Clu’s large “M” domain that is predicted to be unstructured but does not contain canonical intrinsically disordered motifs. It could still be important for particle dynamics, but this has yet to be tested. We previously demonstrated that Clu can self-associate, but we do not yet know whether Clu forms dimers, multimers, or self-association is due to protein aggregation in bliss particles (Sen & Cox, 2016). Determining which domains are required for self-association using ectopic Clu expression has proved challenging in vivo since endogenous Clu must be absent and clu null mutants are quite sick. We have identified that the DUF, Clu, and TPR domains are required for particle assembly and for association with endogenous bliss particles but we do not yet know whether they play a role in molecular self-association in Drosophila. The TPR domain of CLUH has been reported to be necessary for CLUH self-association, although it appears that the self-association is not direct, suggesting CLUH forms multimers (Hemono et al., 2022). In addition, we previously showed that, in Drosophila, the TPR domain is essential for mRNA association (Sen & Cox, 2016), which was confirmed to be the case for CLUH (Hemono et al., 2022). Ectopically expressing full length (FL)-Clu at high levels caused bliss particles to disassemble, which differs from the observation of CLUH granules (discussed below (Pla-Martin et al., 2020)). While this could be due to toxicity, it seems unlikely since overexpression of ΔTPR and ΔDUF did not cause endogenous particle disassembly, nor did overexpression of an unrelated protein CPB. An alternative explanation could be that high concentrations of FL-Clu disrupt required stoichiometry for forming bliss particles. Too much functional Clu could disrupt potential liquid-liquid phase separation that may regulate bliss particle assembly and disassembly, or the increased protein could exert a dominant effect by sequestering factors necessary for particle assembly.

Assembly and maintenance of Clu bliss particles require polysomes

As Clu associates with mRNA and ribosomal proteins, we tested the effect of translation inhibitors on assembly and disassembly with and without stress. CHX and PUR treatment are powerful tools to assess how stalled translation affects the dynamics of particles and granules involved in the posttranscriptional regulation of mRNA. PUR decreases the amount of polysomes, terminating translation, releasing ribosomes, and increasing the amount of messenger RNPs (mRNPs). In contrast, CHX prevents elongation. This results in an inhibition of polysome to mRNP conversion and thus increases stalled ribosomes and decreases levels of mRNPs. Stress granules are composed of mRNPs, stalled preinitiation complexes, and other proteins involved in translation. Under normal conditions, low concentrations of puromycin rarely assemble stress granules, but longer incubation with higher concentrations does (Bounedjah et al., 2014; Ihn et al., 2024; Kedersha et al., 2000; Martinez et al., 2016). In the presence of stress, puromycin increases the number of stress granules, whereas cycloheximide disassembles them. This dynamic is caused by an increased number of mRNPs available with puromycin and a decreased number available with cycloheximide. P-bodies generally contain proteins that are associated with mRNA decay or silencing. P- body maintenance depends on the presence of translationally repressed mRNA. In the presence of CHX, mRNA trapped in polysomes causes P-body loss. However, with PUR treatment, the number and size of P-bodies increase due to the increase in non-translatable mRNPs (Eulalio et al., 2007; Patel et al., 2016).

By treating follicles with PUR and CHX, we found that the availability of polysomes regulates assembly and disassembly of bliss particles. Administering PUR ex vivo rapidly disassembled bliss particles, whereas CHX had no effect ex vivo and in vivo. This suggests that increasing the amount of mRNPs and decreasing polysomes disassembles bliss particles. When particles are absent, CHX treatment blocked insulin- induced assembly ex vivo suggesting that increased polysomes alone are insufficient for Clu particle assembly. The increased polysomes might be physically prevented from separating into Clu particles despite the unchanged Clu levels. This also suggests that insulin signaling, which initiates a strong signaling cascade, cannot overcome the CHX block in assembly. Nutritional and oxidative stress disassemble Clu bliss particles. Both stressors cause decreased translation rates through integrated stress response signaling. During nutritional stress ex vivo and in vivo, CHX treatment maintained Clu particle assembly, suggesting that, once particles are formed, polysomes present in Clu particles stabilize the particles. This was also true for oxidative damage ex vivo caused by hydrogen peroxide. Together, these data support a model whereby Drosophila Clu bliss particles harbor actively translating mRNAs and particle assembly relies on the presence of polysomes. Evidence for Clu bliss particles as sites of active translation is supported by observations of CLUH granules (Pla-Martin et al., 2020).

Comparing Drosophila Clu and vertebrate CLUH subcellular localization

Clu forms large, prominent, and highly dynamic particles in germ cell cytoplasm (Cox & Spradling, 2009; Sheard et al., 2020). These particles are found in Drosophila somatic tissues as well (Sen et al., 2013; Sheard et al., 2020; Wang et al., 2015). We previously demonstrated that Clu particles are highly sensitive to stress. Using live-imaging, Clu bliss particles quickly disassemble in the presence of hydrogen peroxide, and for germ cells lacking particles due to starvation, adding insulin to the media causes particle assembly in minutes (Sheard et al., 2020). Nutritional stress in vivo from starvation causes particle disassembly, with no decrease in protein, and particles reassemble upon feeding (Sheard et al., 2020). Various studies in vertebrate systems have demonstrated CLUH localization in the cell. In COS7 cells, CLUH exhibits a granular pattern, particularly after Triton-X 100 extraction (Gao et al., 2014). In primary hepatocytes and HeLa cells, CLUH is reported to form granules that colocalize with some, but not all, components of stress granules, and these granules increase with stress (Pla-Martin et al., 2020). In addition, in contrast to the data shown here, CLUH overexpression induced the formation of peripheral CLUH granules in about 40% of transfected HeLa cells (Pla-Martin et al., 2020). However, additional reports have shown CLUH is broadly diffuse in the cytoplasm in HCT116 cells and CLUH does not localize with the stress granule component G3BP1 (Hemono et al., 2022). In agreement with our observation, CLUH granules did not disassemble in response to CHX treatment, although these granules were the peripheral granules assembled by overexpression of CLUH (Pla-Martin et al., 2020). Finally, two groups have shown CLUH colocalizes with one or two structures composed of SPAG5/Astrin, which is a mitotic spindle protein during mitosis that localizes to microtubule plus-ends in the cytoplasm during interphase (Dunsch et al., 2011; Hemono et al., 2022; Schatton et al., 2022). It is not clear at present why insects’ Clu localization and dynamics are so different from vertebrate CLUH. One possibility could be that vertebrates may simply have different CLUH dynamics from Drosophila Clu due to differences in cell types and species. Another possibility could be that Clu particles may act differently in vivo compared to CLUH in cell culture experiments due to differences in cell physiology.

Nurse cells with CHX-stabilized Clu particles have reduced cellular stress as indicated by mitochondrial localization

Starvation causes germ cell mitochondria to cluster (Sheard et al., 2020). This ccurs not only with nutritional stress and appears to be a hallmark of cellular stress. When bliss particles are absent, mitochondria are clumped, but when the particles are present, mitochondria disperse throughout the germ cell cytoplasm in the normal pattern. This indicates that normal mitochondrial localization highly correlates with the presence of bliss particles (Cox & Spradling, 2009; Sen et al., 2015; Sheard et al., 2020). We found similar mitochondrial dynamics with CHX treatment. Females fed a rich CHX diet maintained assembled bliss particles and mitochondrial distribution (Fig 5D-F”). Bliss particles that remained assembled with CHX treatment followed by starvation also had normal mitochondrial localization (Fig 6C-E”). This could be attributed to CHX increasing the level of amino acids and thus stimulating TOR activity, a downstream component of the insulin signaling pathway (Beugnet et al., 2003). In addition, Clu particle association with mitochondria decreased particle speed. However, we are unable to distinguish whether the relationship between particle speed and mitochondrial association is because particles physically slow due to the complex size, or whether mitochondria can only associate with slower particles. We were unable to determine whether particle-mitochondrion association affected mitochondrial activity at the single organelle level using TMRE due to variability and efficiency of staining.

In this study, we identified three protein domains of Clu that are necessary to assemble Clu bliss particles. We also found that overexpression of functional FL-Clu causes particle disassembly. In addition, we found that Clu bliss particles require the presence of polysomes to remain assembled and that stabilizing polysomes protects bliss particles from disassembly. Since Clu associates with ribosomes and is a ribonucleoprotein, this supports a model whereby Clu bliss particles are active sites of translation under non-stressed conditions but disassemble when cellular stress increases. Since Clu is closely tied to mitochondrial function and binds mRNAs encoding mitochondrial proteins, particle dynamics likely affects mitochondrial function. Disassembly of particles could lead to decreased translation of the associated mRNAs, fewer mitochondrial proteins actively translated in the cytoplasm, and a shift in metabolism in response to stress. There is evidence supporting this idea through studies on CLUH (Pla-Martin et al., 2020). For a better understanding of Drosophila bliss particles, several challenges must be overcome. There are many proteins known to associate with P-bodies and stress granules (Anderson & Kedersha, 2006; Ivanov et al., 2019). While we and others have identified Clu/CLUH-associated proteins using coimmunoprecipitation and mass spectrometry, we have yet to localize any of them to bliss particles. In addition, we have yet to determine whether bliss particles are active sites of translation. Particles are easily seen in the nurse cells, yet using fluorescent in situ hybridization is challenging. Nonetheless, given Clu’s critical role in mitochondrial function, fully understanding how these unique and novel RNP particles function will ultimately deepen our knowledge of how mitochondria respond to stress.

Fly stocks

Fly stocks were maintained on standard cornmeal fly media. Animals were grown at room temperature. The following stocks were used for experiments: w¹¹¹⁸, w¹¹¹⁸; clu^CA06604 (Cox & Spradling, 2009), w*; clu^CA06604/CyO; nanosGAL4/TM3 Sb, w¹¹¹⁸; UASp-cluΔDUF::mScarlet/TM3 Sb, w¹¹¹⁸; UASp-cluΔClu::mScarlet/TM3 Sb, w¹¹¹⁸; UASp-cluΔTPR::mScarlet/TM3 Sb, w¹¹¹⁸; UASp-FLclu::mScarlet/TM3 Sb, w*; Kr^{If- 1}/CyO; P{w[+mW.hs]=GAL4-da.G32}UH1 (Bloomington Drosophila Stock Center (BDSC), Bloomington, IN, USA, BSC# 55850), and w*; M{w[+mC]=UASp-mCherry.cpb}ZH-86Fb/TM3 Sb¹ (BDSC, Bloomington, IN, USA, BSC# 58728). A newly eclosed fly is considered day 0.

Transgenic flies and constructs

For the C-terminal fusion of mScarlet for live-imaging, we created a Gateway destination vector pPgateWmScarlet-i for subcloning. Briefly, the mScarlet-i coding sequence was amplified from pCytERM_mScarlet-i_N1 (Addgene, Watertown, MA, USA, cat#. 85068) using the following primers, 5’-TAG GCC ACT AGT GTG AGC AAG GGC GAG GCA GT-3’and 5’-TGC TTA GGA TCC TTA CTT GTA CAG CTC GTC CA-3’. The amplicons were subcloned into a pQUASp (Addgene, Watertown, MA, USA, cat#. 46162), placing the UASp promoter upstream of the mScarlet-i coding sequence. Ampicillin resistance was used to select positive clones, which were verified by restriction digest and sequencing. The resulting pUASp-mScarlet-i construct was converted to a Gateway destination vector using the Gateway^™ Vector Conversion System (Invitrogen, Waltham, MA USA, cat#. 11828029). Chloramphenicol resistance was used to select positive clones, which were verified by sequencing. The pQUASp vector was a gift from Christopher Potter (Addgene plasmid #46162; http://n2tnet/addgene:46162; RRID: Addgene_46162), and pCytERM_mScarlet-i_N1 vector was a gift from Dorus Gadella (Addgene plasmid # 85068; http://n2t.net/addgene:85068; RRID: Addgene_85068) (Bindels et al., 2017). Gateway entry vectors with full-length Clu (Clu_pENTR) or domain-deleted Clu (ΔDUF_pENTR, ΔClu_pENTR, and ΔTPR_pENTR) were previously described (Sen & Cox, 2016). Each entry vector was cloned into pPgateWmScarlet-i using LR Clonase mix (Invitrogen, Waltham, MA, USA, cat#. 11791020) following the manufacturer’s directions. The resulting expression vectors were selected by ampicillin resistance and verified by sequencing. For transgenic flies, the vectors were commercially injected (BestGene Inc. Chino Hills, CA, USA).

Live-imaging for analysis of Clu particle dynamics

Live-imaging with ovarioles was performed as previously described (Sheard et al., 2020) with some modifications. Ovaries were dissected in a live-imaging media composed of Complete Schneider’s (CS) media and 200 μg/mL of insulin. The CS media was Schneider’s Drosophila medium (Fisher Scientific, Hampton, NH, USA, cat#. BW04351Q) supplemented with 15% heat-inactivated fetal bovine serum (CPS Serum, Parkville, MO, USA, cat#. FBS-500HI) and penicillin (100 U/mL) - streptomycin (100 μg/mL) (Fisher Scientific, Hampton, NH, USA, cat#. BW17602E). After separating ovarioles from an ovary and eliminating the connected muscle sheath and nerve fibers, the tissues were transferred into a 35 mm MatTek glass bottom dish (MatTek Corporation, Ashland, MA, USA, cat#. P35G-0-20-C) with a live-imaging media. To simultaneously visualize Clu particles and mitochondria, TMRE (tetramethylrhodamine, ethyl ester, perchlorate) (Anaspec Inc, Fremont, CA, USA, cat#. AS88061) was diluted to 50 nM in the dissection media. Tissues were incubated for 20 minutes and imaged without washing. The follicles were mainly chosen between stages 5 to 7, previtellogenic stages, for better observation with nurse cells. The focal plane was selected to have at least three to four nurse cells with a clear visibility of nuclear and cytoplasmic areas, with appproximately 25% depth from the top surface of a follicle. Follicle stages were determined with a size of a follicle as described in the reference (Spradling, 1993). More details for each experiment, including the number of dissected animals and replicates are describe in supplementary tables. Live images were obtained using a Nikon A1 plus Piezo Z Drive Confocal microscope at 60x (Nikon Corporation, Tokyo, Japan) or a Nikon Eclipse Ti2 spinning disk microscope at 100x (Nikon Corporation, Tokyo, Japan). Fiji ImageJ was utilized to analyze confocal images (Schindelin et al., 2012).

Live-imaging for Clu particle dynamics with puromycin, cycloheximide and hydrogen peroxide treatment

The working solution for each chemical was prepared just before performing an experiment as follows: 10 mg/mL puromycin (Gibco^™, Waltham, MA, USA, cat#. 1113803) was diluted to 20 μM in a media composed of Complete Schneider’s (CS) media and 100 μg/mL of insulin (CS/Ins); cycloheximide powder (CHX, Sigma-Aldrich, Burlington, MA, USA, cat#. C7698) was dissolved in CS for 14 mM stock solution, which was further diluted to 3.5 mM and 7 mM CHX-containing CS or CS/Ins media; and 30% hydrogen peroxide (Sigma-Aldrich, Burlington, MA, USA, cat#. H1009) was diluted to 4% hydrogen peroxide in CS/Ins or CHX-containing CS/Ins. Ovaries were dissected as described above with corresponding media, CS or CS/Ins, depending on the purpose of each experiment. To test the puromycin effect on bliss particle dynamics, dissected ovaries with CS/Ins media were transferred into a 35 mm MatTek glass bottom dish with 50 μL of CS/Ins media, and live imaging was performed after adding 50 μL of CS/Ins containing 20 μM puromycin to the ovaries to make a final concentration of 10 μM puromycin. To test the CHX effect on bliss particle dispersion, dissected ovaries with CS/Ins media were incubated for 20 minutes with 3.5 mM CHX-containing CS/Ins (CS/Ins/CHX) following twice wash with the same media, and then a live-imaging was performed. To test the CHX effect on bliss particle formation, ovaries were dissected from starved animals in CS, incubated for 20 minutes with 3.5 mM CHX-containing CS (CS/CHX) following twice wash with the same media, re-washed twice with CS/CHX containing 100 μg/mL insulin (CS/CHX/Ins), and then switched to CS/CHX/Ins for immediate live-imaging. To test the CHX effect on particle dispersion without insulin, ovaries were dissected from w¹¹¹⁸; clu^CA06604 in CS, washed twice with CS containing 3.5 mM CHX (CS/CHX), incubated for 30 minutes with the same media, and then live images were obtained. To produce oxidative stress, dissected ovaries were incubated with 50 μL of CS/Ins or CS/CHX/Ins for 20 minutes following twice wash with a corresponding media. Live-imaging was performed after adding 50 μL of each corresponding media containing 4 mM hydrogen peroxide to make a final concentration of 2 mM hydrogen peroxide. Images were obtained using a Nikon A1plus Piezo Z Drive Confocal microscope at 60x or a Nikon Eclipse Ti2 spinning disk microscope at 100x. Selection of follicle stages and a focal plane was performed as described above. More details of each experiment including the number of dissected animals and replicates are described in S2 Table.

Generating and analysis of kymographs

The still frames of the time-lapse image were stacked to track particle movements. Fiji ImageJ was utilized to generate kymograph and measure velocity with a tool, Multi Kymograph, or Plugins, KymographBuilder (Mary et al., 2016; Schindelin et al., 2012). To measure the velocity of directed Clu particles for CHX treatment, live time-laps images were recorded for 3 minutes and analyzed as follows. Six areas within a follicle were chosen randomly, as shown in Fig 8B, applied with a segmented line to generate a kymograph. A clu particle showing a progressive movement within a kymograph was chosen, and velocity was calculated (more details in S2 Table). To measure the speed of Clu particles of various sizes in relation to mitochondrial association, live time-laps images were recorded for more than 3 minutes after TMRE staining as described above. Clu particle that could be tracked for 1 minute was selected, and the movement of each particle was traced with a segmented line to generate kymograph, which was used to calculate speed (more details in S3 Table). Clu particle-mitochondrial binding was judged by juxtaposing the representative color of Clu (green) and mitochondria (magenta). The size of each Clu particle was calculated using ROI as the area of each particle. Using GraphPad Prism, the graphs were generated and the data was analyzed statistically with unpaired t-test, calculation of Pearson correlation coefficient, and logistic regression (GraphPad Software, Boston, Massachusetts USA, www.graphpad.com).

Preparation of cycloheximide solution for wet yeast paste to feed flies

CHX was dissolved in water to prepare a 10 mM stock solution that was aliquoted and stored at −20 °C until use. The desired concentration of CHX was prepared by serial dilution of the stock solution. 0.3 g of active dry yeast powder (Red Star^® Yeast) was mixed with 450 μL of CHX solution to create a yeast paste which was provided daily as needed.

Cycloheximide feeding for ovary analysis

Ten female day 0 adult flies and ten male day 0 adult flies were collected in a standard food vial with wet yeast paste, and on day 3, female adults were separated from males. The food vial with fresh wet yeast paste was switched every day to make flies fatten until day 4. On day 4, all female flies in each vial were transferred to a standard food vial containing freshly made yeast paste with CHX. In 24 hours after CHX feeding, fly ovaries were dissected in Grace’s insect media. To determine the effect of starvation after CHX feeding, after feeding CHX for 24 hours, the flies were transferred to an empty vial with a wet piece of tissue with water and maintained for 5 hours. The flies were then dissected and immunostained. The numbers of dissected animals and replicates are described in S2 Table.

Immunostaining

Ovaries were dissected with Grace’s Insect Medium (Invitrogen, Waltham, MA, USA, cat#11595030) and fixed for 20 minutes (4% paraformaldehyde and 20 mM formic acid in Grace’s Insect Medium). After washing with Antibody wash solution (AWS, 0.1% Triton X-100 and 1% BSA in phosphate-buffered saline) three times for twenty minutes, the tissue was stained with primary antibody overnight at 4 °C. After washing with AWS three times for twenty minutes, the tissues were stained with secondary antibodies overnight at 4 °C, then washed with AWS twice for twenty minutes and stained with 5 ng/mL 4',6-Diamidino-2-phenylindole (DAPI) solution for ten minutes. After removing the DAPI solution, the tissues were mounted in Vectashield Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA, cat#. H-1000). Images were obtained using a Zeiss LSM 980 confocal laser scanning microscope (Carl Zeiss Microscopy LLC, White Plains, NY, USA). The follicles were mainly chosen between stages 5 to 7, previtellogenic stages, for better observation in nurse cells. The focal plane was selected for ensuring at least three to four nuclei were clearly visible in a nurse cells, with approximately 25% depth from the top surface of a follicle but also to avoid a dim fluorescence signal due to a deeper depth for a fixed imaging. The numbers of dissected animals and replicates are described in supplementary tables. The following antibodies were used: guinea pig anti-Clu N-terminus (1:2000 (Cox & Spradling, 2009)), rat anti-mScarlet-i sdAb-FluoTag-X2 (1:1000, Synaptic System, Goettingen, Germany, cat#. N1302-At488-L), chicken anti-mCherry (1:1000, Novus Biologicals, Centennial, CO, USA, cat#. NBP2-25158), mouse anti-ATP5A1 (1:1000, Abcam, Cambridge, UK, cat#. 14748, or Invitrogen, Waltham, MA, USA, cat#. 439800), goat anti-guinea pig Alexa 488 (1:1000, Invitrogen, Waltham, MA, USA, cat#. A11073), goat anti-guinea pig Alexa 633 (1:1000, Invitrogen, Waltham, MA, USA, cat#. A21105), goat anti-chicken Alexa 568 (1:500, Invitrogen, Waltham, MA, USA, cat#. A11041), goat anti-mouse IgG2b Alexa 488 (1: 500, Invitrogen, Waltham, MA, USA, cat#. A21141), goat anti-mouse IgG2b Alexa 568 (1: 500, Invitrogen, Waltham, MA, USA, cat#. A21144).