Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in  To reduce chromosome number from diploid to haploid during sexual reproduction, homologous chromosomes must segregate to different daughter cells in the first division of meiosis. Most organisms achieve this segregation by linking homologous chromosomes with chiasmata, exchanges of continuity between chromatids that derive from repair of programmed double-strand breaks (DSBs). DSBs are created by the conserved endonuclease Spo11 acting in concert with an array of cofactors . The iniDrosophila  that holds chromosomes together in meiosis. This dependence has led to the conclusion that synapsis between homologous chromosomes is dependent on successful meiotic recombination in these organisms . In contbination , and thubination . The extC. elegans (PPH-4.1) is required for viability-supporting levels of DSB initiation as well as homologous pairing and synapsis during meiotic prophase (Arabidopsis (Protein substrates phosphorylated by ATM and ATR are known to be dephosphorylated in many contexts by the highly conserved serine/threonine protein phosphatase 4 (PP4) . Previouprophase . PP4 hasprophase  and suppbidopsis .C. elegans ATL-1): meiotic DSB levels are decreased by the phosphorylation of DSB-1, but drastically increase when DSB-1 cannot be phosphorylated. During meiotic prophase, DSB-1 is phosphorylated in an ATL-1-dependent manner to inhibit DSB formation and protect the genome against excessive DSBs. In contrast, DSB-1 is dephosphorylated in a PPH-4.1-dependent manner, thereby promoting a number of DSBs sufficient to form a crossover on each chromosome pair and allow proper chromosome segregation. Since ATM/ATR kinases are known to be activated by DNA breaks, our model predicts that activated ATR kinase turns off the DSB machinery via DSB-1 phosphorylation once sufficient levels of recombination intermediates are generated. This feedback mechanism could tune DSB levels to ensure the formation of crossovers via phosphoregulation of DSB-1. Moreover, we find that the homologous pairing and synapsis defects in pph-4.1 mutants are significantly rescued when DSB levels are increased by a non-phosphorylatable allele of dsb-1, adding to the growing evidence that DSBs can strengthen homologous synapsis in C. elegans. Our results shed light on fundamental mechanisms of meiotic chromosome dynamics regulated by contrasting kinase and phosphatase activities.In this work, we show that the DSB-promoting activity of DSB-1 is controlled by both PPH-4.1 and ATR  generation. The effectiveness of pph-4 RNAi was verified by observing univalents in diakinesis oocytes . In extracts from the pph-4.1 RNAi-treated animals, the proportion of the upper band was significantly stronger than the band at the expected size . This result suggests that depletion of PPH-4.1 leads to hyperphosphorylation of DSB-1. To test whether our observations were influenced by the fusion of GFP to DSB-1 protein, we next performed western blots using polyclonal antibodies directed against DSB-1 itself. Since pph-4.1 mutants are mostly embryonic inviable, in order to obtain sufficient pph-4.1-deficient material, we made lysates from a mixed population of pph-4.1 homozygous and balanced heterozygous mutant adults further treated with pph-4.1 RNAi, and obtained a similar shift compared to a wild-type control , htp-3(tm3655), chk-2(me64), rad-50(ok197), mre-11(ok179)) . In other words, in mre-11 and rad-50 mutants, DSB-1 was not phosphorylated, or was phosphorylated very weakly, even in the presence of DSBs. This is consistent with the previous observation that the MRN (Mre11/Rad50/Nbs1) complex plays an important role in activating ATM/ATR kinases ) . In all  mutants , D. In o kinases . These dpph-4.1(tm1598) mutation with the atm-1 or atl-1 mutations. We assessed DSB formation in these double mutants by performing immunofluorescence against the strand-exchange protein RAD-51. Since the C. elegans gonad contains nuclei from all stages of meiotic prophase arranged sequentially along the distal-proximal axis, we divided the gonad into seven zones of equal size (pph-4.1 led to a drastic decrease of the number of foci compared to wild-type controls (atm-1 in C. elegans) in a pph-4.1 background only marginally increased the number of DSBs in very late pachytene. Since homozygous mutation of ATR (atl-1 in C. elegans) leads to severe mitotic defects and aneuploidy in the germline due to replication errors (atl-1 in a pph-4.1 mutant led to a strong recovery of RAD-51 foci (atl-1(tm853) mutation led to a significant increase in overall DSB number in the presence of wild-type pph-4.1  in a rad-54(ok615) mutant background, in which DSBs are initiated but RAD-51 cannot be removed from recombination intermediates , in which all five SQ serines were replaced by alanine (dsb-1(5A); pph-4.1 animals to assess DSB formation. Germlines homozygous for both pph-4.1 and dsb-1(5A) showed a drastically higher number of RAD-51 foci compared to pph-4.1 single mutants (dsb-1(5A) alone showed significantly elevated DSB levels compared to wild-type controls, suggesting that phosphorylation of these serine residues acts in wild-type germlines to limit the number of DSB initiations. We noted that the appearance of RAD-51 foci peaks is delayed in pph-4.1 and dsb-1(5A); pph-4.1, as well as in pph-4.1; atl-1/nT1 (pph-4.1 mutants at the 24 hr post-L4 stage (DSB-1 possesses five ATM/ATR consensus motifs ([ST]Q), two of which are highly positionally conserved in the genus habditis . These Shabditis . Moreovehabditis ; all DSBhabditis . The pre alanine . This no alanine . We then mutants . Indeed,tl-1/nT1 . Previoutl-1/nT1 . DSB reptl-1/nT1 . We haveL4 stage . Since Pdsb-1(5A); pph-4.1 double mutants. Since meiotic nondisjunction of the X chromosome leads to production of males (with a single X chromosome) among the self-progeny of C. elegans hermaphrodites, we examined the frequency of male progeny in pph-4.1; dsb-1(5A) mutants, and found that it decreased significantly compared to pph-4.1 single mutants . While pph-4.1 single mutants have a very low embryonic viability of 2% on average, the viability of pph-4.1; dsb-1(5A) double mutants increased to 41% . It is therefore likely that these phosphomimetic mutations do not functionally simulate the phosphorylation of DSB-1.To test whether constitutively phosphorylated DSB-1 results in a decreased number of DSBs, we constructed dsb-1(5A) mutants, dsb-2 non-phosphorylatable mutants, (dsb-2(S110A_S116A_S143A_S167A), hereafter called dsb-2(4A)) in which all four SQ serines were replaced by alanine, did not show increased RAD-51 foci compared to the wild type, suggesting that DSB-2 is refractory to phosphoregulation  in its predicted disordered region. In contrast to gulation .pph-4.1; dsb-1(5A) animals was somewhat surprising, given our previous finding that autosomal pairing is severely reduced in pph-4.1 single mutants  mutants might promote bivalent formation by increasing the level of homologous pairing. To test this hypothesis, we carried out FISH against the 5S rDNA locus on chromosome V to assess the progression of homologous pairing over time, in pph-4.1 compared to pph-4.1; dsb-1(5A) mutants. While the pph-4.1 mutant showed very low homologous pairing as previously shown  double mutant rescued pairing to a significant degree, up to 70%, and the dsb-1(5A) single mutant showed wild-type timing and levels of homologous pairing  double mutants compared to pph-4.1 single mutants (pph-4.1 mutation leads to non-homologous synapsis including both fold-over synapsis within a single chromosome and promiscuous synapsis between non-homologous chromosomes (dsb-1(5A) allele improves homologous synapsis of pph-4.1 mutants by changing the timing of synapsis initiation or completion, we performed immunofluorescence against the SC axial element protein HTP-3 and the central region protein SYP-2 in whole gonads of wild type, pph-4.1, and pph-4.1; dsb-1(5A), and found no difference in the timing of synapsis  animals by counting the number of DAPI-stained bodies in oocytes at diakinesis  mutants was significantly higher than in pph-4.1 single mutants.We verified the formation of bivalents in akinesis . In the  mutants . As expepph-4.1; dsb-1(5A) mutants contrasts with our previous observation that γ-ray irradiation (10 Gy) did not rescue bivalent formation in pph-4.1 mutants  mutants, although 50 Gy of γ-rays leads to more RAD-51 foci than the 5A allele (data not shown). Since 10 Gy irradiation at least partially rescues bivalent formation in mutants carrying the null spo-11(me44) allele, but does not rescue pph-4.1 mutants  null mutants, a profound reduction of DSBs leads to crossover defects, causing severe embryonic inviability that increases with maternal age  allele to rescue the loss of DSBs in pph-4.1 mutants suggests that the 5A allele is hyperactive and not subject to downregulation through phosphorylation. To test whether this hyperactive allele depends on DSB-2 for its high levels of break formation, we performed RAD-51 immunofluorescence on dsb-1(5A); dsb-2 germlines. In agreement with previous observations, we found very few RAD-51 foci in dsb-2 mutant nuclei. However, the dsb-1(5A) allele strongly rescued DSB formation in the dsb-2 null background to levels higher than wild type, similar to what is seen in the dsb-1(5A) allele alone  double mutants , which is dsb-1(S186A); dsb-1(2A), which is dsb-1(S137A_S143A) and dsb-1(3A), which is dsb-1(S137A_S143A_S186A) based on the observation that S137 and S186 are highly conserved within the elegans group of Caenorhabditis (dsb-2 mutants. We found that the both dsb-1(1A) and dsb-1(3A) alleles were sufficient to rescue embryonic viability to wild-type levels at all maternal ages (dsb-1(2A) mutation, which does not include S186, rescued the embryonic viability defect in young dsb-2 adults , but the rescue was less pronounced in older animals (day 3 post-L4) . We therefore conclude that phosphorylation of any of the SQ motifs in the intrinsically disordered region of DSB-1 may contribute to shutting down the DSB-promoting activity of DSB-1, with S186 phosphorylation likely to be a strong determinant of reduced DSB activity in aged animals. Consistent with this, quantification of RAD-51 foci in dsb-1 (1A), (2A), (3A), and (5A) mutants revealed that the mutants containing S186A  showed an increase in DSB levels either in early prophase (zone 3 for 1A and 3A) or throughout prophase (for 5A) compared to the wild type  , left. Wild type . We alsoild type .dsb-1(1A) allele fully rescued dsb-2 mutants, it did not rescue embryonic viability in pph-4.1 mutants (dsb-1(1A) mutants showed mildly increased levels of RAD-51 foci compared to wild type, and introducing the dsb-1(1A) mutation increased RAD-51 foci both in pph-4.1; dsb-1(1A) and in dsb-2; dsb-1(1A) mutants compared to the respective single pph-4.1 or dsb-2 mutants  mutants, in which DSBs are generated but the resulting recombination intermediates are trapped without completion of repair (gfp-dsb-1 or gfp-dsb-1(5A) worms in varying combination with dsb-2 and 24 hr treatment with pph-4.1 RNAi, blotted onto membranes and probed with α-GFP antibodies. In gfp-dsb-1 animals, the proportion of a slow-migrating band significantly increased in older animals  animals, the phosphorylated band proportion is strongly reduced both in young and old animals compared to the control. While the slow-migrating band is greatly reduced, a smearing of GFP-DSB-1(5A) can be seen above the main band (We and others have shown that DSB initiation activity is significantly reduced in older adults (over 72 hr post-L4) compared to young adults (24 hr post-L4) in f repair . We ther animals , demonst studies . The pho animals . Howeverain band . DSB-1 idsb-3 as a nematode ortholog of Mei4 and its likely participation in a complex with DSB-1 and DSB-2  promotes Spo11 activity remains unknown, how phosphorylation of these factors negates their activity is also not clear. Recent work has shown that neutralizing a conserved basic patch of either Rec114 or Mer2 leads to loss of DNA binding ; analogoCaenorhabditis. Based on our inferred gene tree, we estimate that the C. elegans DSB-1 and DSB-2 protein sequences have undergone 1.36 and 2.02 amino acid substitutions per site, respectively, since the gene duplication event  mutant rescues loss of dsb-2. These results suggest that DSB-2 is required to counteract the phosphorylation and thus downregulation of DSB-1. Taken together with previous results showing that loss of dsb-2 leads to reduction in DSB-1 protein levels  allele as well as γ-irradiation can increase homologous pairing, synapsis, and bivalent formation in pph-4.1 mutants  correction of synapsis does. Interestingly, the 5A allele rescues pph-4.1 to a greater degree than 50 Gy of γ-rays, even though the number of induced breaks in γ-irradiated pph-4.1 worms is higher than that seen in pph-4.1; dsb-1(5A) (data not shown). This difference has several possible explanations: perhaps the mechanism that corrects promiscuous synapsis requires canonical SPO-11-catalyzed breaks rather than the more complex damage caused by γ-rays. Alternatively, hyperactivation of the DNA damage response by high levels of both single-strand break and DSB in γ-irradiated worms may not allow homologous synapsis to reach the level seen in pph-4.1; dsb-1(5A) animals, or may interfere with efficient conversion of recombination intermediates to COs. In a similar vein, γ-ray-induced desynapsis  allele suffices to fully rescue mutations in dsb-2, but has little effect on pph-4.1  mutants  germlines show numbers of RAD-51 foci intermediate between pph-4.1 and pph-4.1; dsb-1(5A)  double mutants  , we hypooduction  that are mutants .dsb-1(5A) mutation enjoy full viability, with brood size and male incidence nearly identical to wild type despite the roughly twofold higher number of RAD-51 foci observed, raises the question of what role negative control of DSBs is playing in C. elegans. To examine whether dsb-1(5A) could sensitize worms to external DNA damage, we have γ-irradiated control and dsb-1(5A) animals and assayed for embryonic inviability as an indicator of unrepaired DNA breaks. However, dsb-1(5A) mutants showed no difference from control animals in embryonic viability or brood size after 30 or 75 Gy irradiation (data not shown), suggesting that meiocytes have a large capacity to repair DSBs in excess over wild-type levels. A recent study examining the effects of loss of germline ATM in mice  plates seeded with OP50 bacteria under standard conditions  and dsb-1 non-phosphorylatable lines. A 10 µL mixture containing 17.5 µM trans-activating CRISPR RNA (tracrRNA)/crRNA oligonucleotides (targeting dsb-1 and dpy-10) purchased from Integrated DNA Technologies , 17.5 µM Cas9 protein produced by the MacroLab at UC Berkeley, and 6 µM single-stranded DNA oligonucleotide purchased from IDT or 150 ng/µL double-stranded DNA generated from PCR as a repair template was injected into the gonads of 24 hr post-L4 larval stage N2 hermaphrodites. To prevent re-editing by the CRISPR-Cas9 machinery, silent mutations were introduced into the target gene dsb-1. For gfp-dsb-1, an additional linker sequence of 3× glycine was introduced between the target site and GFP-tag sequence. Dpy or Rol F1 animals  were picked to individual plates to self-propagate overnight and then screened for successful edits by PCR and DNA sequencing. A list of oligonucleotides used is provided in CRISPR-Cas9 genome editing using R marker  was applgfp-dsb-1 worms with the HT115 bacteria expressing either the empty RNAi vector L4440 obtained from the Ahringer Lab RNAi library +0.01% Tween buffer, washed three times with M9 buffer and distributed to RNAi plates. About 30 hr later, the worms became gravid and were harvested in M9 + 0.01% Tween buffer, washed three times with M9 buffer and bleached for no more than 3 min to obtain F1 embryos. Collected embryos were placed to fresh RNAi plates and grown until 24 hr after the L4 larval stage. For western blot analysis, worms were harvested and washed three times or more in M9 buffer and frozen in liquid nitrogen.RNA interference (RNAi) was carried out by feeding N2 or  library  or a pph plasmid . Worms wC. elegans germline was performed as previously described  were used as a control. To obtain synchronized worms, L4 hermaphrodites were picked from the maintenance plates. Auxin treatment was performed by transferring worms to auxin plates and incubating for the indicated time at 20°C. Young adult animals (24 hr post-L4) were dissected for immunofluorescence analyses.Depletion of AID-tagged proteins in the escribed . Brieflyg at 4°C for 15 min. The supernatant was used to measure protein concentration using the BCA kit , and a set amount of protein was loaded for western blotting after boiling for 10 min in SDS-PAGE sample buffer.To prepare samples for general western blotting of GFP-DSB-1, frozen worm pellet was suspended in urea lysis buffer , sonicated  and spun down at 16,000 gfp-dsb-1 worms treated with pph-4 RNAi were suspended in lysis buffer  containing protease inhibitor cocktail  and sonicated . This lysate was first spun down for 100 g at 4°C for 3 min to remove worm debris. The supernatant was further spun down at 16,000 g at 4°C for 15 min to pellet nuclei, and the pellet was resuspended in RIPA buffer , 50 mM Tris pH 8.0) containing protease inhibitor cocktail  and incubated at 4°C for 30 min to solubilize nuclear proteins. The lysate was further sonicated  and used for the phosphatase assay  following the manufacturer’s instructions at 30°C for 2 hr. Then the SDS-PAGE sample buffer was added to the phosphatase reaction, boiled at 95°C for 10 min and used for western blotting.For the phosphatase assay, frozen For endogenous DSB-1 immunoblotting experiments in For western blotting of GFP-fused DSB-1, SDS-PAGE was carried out using 5–12% Wako gradient gel , and proteins were transferred to a PVDF membrane at 4°C, 80 V for 2.5 hr. The membrane was blocked with TBST buffer (TBS and 0.1% Tween) containing 5% skim milk  at room temperature for 1 hr and probed with primary antibody solution containing 2.5% skim milk at 4°C overnight followed by additional 2 hr at room temperature, washed with TBST for four times, probed with secondary antibody solution containing 2.5% skim milk at room temperature for 2 hr, washed with TBST for four times. Chemi Luminol assay kit, Chemilumi-one super , or Chemilumi-one ultra  was used to visualize protein bands using an ImageQuant LAS4000 imager .For endogenous DSB-1 immunoblotting experiments, gel electrophoresis was performed using 4–12% Novex NuPage gels . Proteins were transferred to a PVDF membrane at 4°C, 80 V for 2.5 hr. The membrane was blocked at room temperature for 1 hr in TBST containing 5% skim milk and then probed with primary antibody solution containing 5% skim milk at 4°C overnight or at room temperature for 2 hr, washed three times with TBST containing 1% skim milk, probed with secondary antibody solution containing 1% skim milk at room temperature for 2 hr, and washed with TBST for three times before proceeding to detection.2) coverslip.Immunostaining was performed as described in Images were captured by a Deltavision personalDV microscope  equipped with a CoolSNAP ES2 camera (photometrics) at a room temperature of 20–22°C, using a 100× UPlanSApo 1.4NA oil immersion objective  and immersion oil  at a refractive index of 1.513. The Z spacing was 0.2 µm and raw images were subjected to constrained iterative deconvolution followed by chromatic correction. Image acquisition and deconvolution was performed with the softWoRx suite . Image postprocessing for publication was limited to linear intensity scaling and maximum-intensity projection using OMERO .2 coverslip. The worms on the slides were touched to the drop of probe solution on the coverslip until the liquid was spreaded out. After being sealed, the slides were denatured at 95°C for 2 min 10 s and incubated at 37°C overnight. The slides were then washed with 50% formamide in 2× SSCT at 37°C twice for a total of 1 hr, and washed with 2× SSCT for 10 min, stained with DAPI, washed again with 2× SSCT, and mounted with 15 µL mounting medium onto clean Matsunami No. 1S (22 mm2) coverslip. Quantification of FISH foci was done as in The pairing on the right arm of chromosome V was monitored with FISH probes that label the 5S rDNA locus as described in rad-54 and atm-1; rad-54 mutants, manual counting was performed. For rad-54 mutants, semi-automated counting was used as below: for early zones (1 and 2) with very few RAD-51 foci, manual counting was performed. For zones 3 and above, programs  in a Cs-137 Gammacell 40 Exactor . Irradiated worms were fixed 18–22 hr after irradiation for DAPI staining, and imaged to score DAPI-stained bodies as above. For western blotting of DSB-1, approximately 24 hr post-L4 worms were irradiated with either 10 or 100 Gy of γ-rays, and animals were lysed 1 hr post-irradiation.To score embryonic viability and male progeny of each genotype, L4 larval stage hermaphrodites (P0s) were picked individually onto plates and transferred to fresh plates every 24 hr for 5 days. Unhatched eggs remaining on the plates 20 hr after being laid were counted as dead eggs every day. Viable F1 progeny and males were scored 4 days after P0s were removed from corresponding plates.github.com/sokrypton/ColabFold, commit ebf4df8) to the AlphaFold2 pipeline on the Colab platform (Google Research). Prediction was run on trimers using protein sequences for DSB-1, DSB-2, DSB-3 (C. elegans and C. inopinata) retrieved from Wormbase (Homo sapiens and Saccharomyces cerevisiae) retrieved from Uniprot . Due to the high diversity within this group, the list was pared down to DSB-1 orthologs of 11 species in the elegans group  from WormBase ParaSite (Caenorhabditis Genomes Project website (http://caenorhabditis.org/). We used AGAT v0.4.0  and DSB-2 (CELEG.F26H11.6) was inferred using maximum likelihood (LG substitution model with gamma distributed rate variation). The DSB-1/DSB-2 duplication event is denoted by a gray circle. Branch lengths represent the number of substitutions per site; scale is shown at left. The tree is rooted on the branch subtending the Caenorhabditis monodelphis and Caenorhabditis auriculariae sequences.Gene tree of the orthogroup containing the  The connection between double-strand break (DSB) formation and chromosome pairing/synapsis during meiosis is not fully understood. In this paper, the authors show that the formation of DSBs is regulated by the DNA damage response (DDR) machinery. The paper will be of interest to the broad meiosis and DDR communities. 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, including Federico Pelisch as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jessica Tyler as the Senior Editor.Thank you for submitting your article "Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity" 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.Essential revisions:1) For the phosphorylation analysis, the authors should change these qualitative observations to a more quantitative method.2) Clarify the IR dosage selection and make it explicit in each Figure.3) Analysis of SC assembly in pph-4.1; atl-1/nT1 and pph-4.1; dsb-1(5A) mutants.4) Comparison of the levels and kinetics of RAD-51 foci in the dsb-1 phosphomutant series.As an additional point, and this is entirely your choice, all reviewers felt that the AlphaFold analysis adds a mechanistic understanding of the respective roles of DSB1 and DSB-2 and could be moved to the Results section.Reviewer #1 (Recommendations for the authors):Specific points:1. What is the rationale for going straight into DSB-1? While it is a perfectly reasonable target to investigate, an explanation for the non-expert would be beneficial.2. For clarity, what is the length of the RNAi treatments? Along this same line, Figure S1B uses a combination of allele+RNAi. The choice of depletion/loss of function in the different figures should be explicitly explained.3. When mentioning the DSB-1 shifts in Figure S1, the authors greatly simplify these results by stating that only in the mre-11 and rad-50 mutants DSB-1 phosphorylation is absent or significantly reduced. However, htp-3 also seems to lead to significantly reduced DSB-1 phosphorylation. The authors should change these qualitative observations to a more quantitative method (maybe phospho vs non-phospho ratio?), preferably using a detection method with a decent linear range (i.e. LiCor).4. Related to 3, there seems to be a change in DSB-1 levels accompanying the phosphorylation changes. Is there a link between phosphorylation and stability?5. Is the DSB-1 shift lost in the 5A mutant? In fact, an analysis of the different phospho-mutants would be informative.6. I got confused with the different IR doses used. For example, the result in Figure 1D suggests to me that 100 Gy would be the best to analyse the effect of kinase mutants, but in Figure 1E 10 Gy is used. Could this be clarified?7. Also in Figure 1E, it would be interesting to see the effect of the single atl-1 and atm-1 mutants on DSB-1 phosphorylation.8. In Figure 5, the shifts do not seem equal. For example, the shift in the gfp::dsb-1; pph-4.1 seems significantly higher than the other ones. Have the authors tried phos-tag gels to get some insight into the possibility of different DSB-1 phospho-isoforms? At least this should be mentioned to make it clear that this is a complex analysis in spite of analysing through a 'simple' shift in a gel. In this Figure, the difference in total DSB-1 levels in the different conditions is very big, again pointing to the possibility that stability (linked to phosphorylation or not) plays a role.Reviewer #2 (Recommendations for the authors):My specific points are listed below:1. Throughout the paper , the status of DSB-1 phosphorylation was assessed by the band shift in western blot analyses of DSB-1. Quantification of the fast and slow migrating DSB-1 and presenting their ratio from biological replicates will strengthen this claim and show the degree to which PPH-4.1 contributes to DSB-1 dephosphorylation.2. In Figure 1B, please explain in the text why irradiated worms were used for this experiment. While it was shown in Figure 1D that the level of DSB-1 phosphorylation is increased upon irradiation, the context needs to be provided when it was first shown in the paper.3. In Figures 1F-H, the authors tested how ATM-1 or ATL-1 kinases oppose PPH-4.1 activity by comparing the kinetics of RAD-51 loading in various single and double mutant series and showed that reducing the gene dose of atl-1, but not the atm-1 mutation, restores RAD-51 loading of pph-4.1 mutants. Have the authors examined the band shift of DSB-1 in single mutants of atm-1 or atl-1 on western blots? This will directly test whether ATL-1, but not ATM-1, is the major kinase responsible for DSB-1 phosphorylation.4. In Figures 1H and 2C, the kinetics of RAD-51 appearance/disappearance differ between atl-1/nT1 and pph-4.1; atl-1/nT1 and between dsb-1(5A) and pph-4.1; dsb-1(5A). The authors explained this in the text, implicating PPH-4.1 in processing recombination intermediates. Could this also (or rather) reflect the requirement of PPH-4.1 in proper synapsis and thus the delayed meiotic progression in pph-4.1 mutants? Have the authors examined SC assembly in pph-4.1; atl-1/nT1 and pph-4.1; dsb-1(5A) mutants?5. On page 4, in the final sentence of the 1st paragraph, the authors stated, "these results indicate that introduction of DSBs into pph-4.1 mutants also leads to increased fidelity of pairing and synapsis". However, the status of SC assembly was not examined in the current work. Examining the SC structure would be also important in interpreting the rescue results with the dsb-1(1A) in Figure 5, as it will further elucidate the role of PPH-4.1 in other meiotic processes.6. In Figure 3D, since the dose of irradiation is important for this experiment, please indicate that 50Gy of γ-ray was used on the graph.7. When comparing the degree of rescue shown in Figure 3C and 3D, pph-4.1; dsb-1(5A) animals seem to be more proficient in chiasma formation than pph-4.1 γ-irradiated (50 Gy). How do the DSB levels compare between dsb-1(5A) and 50 Gy irradiation? Can this be explained by the level of DSBs induced?8. In the current flow of the paper, I feel that Figure 4 doesn't add much insight into the overall story, as it is somewhat expected . Perhaps, this could be shown earlier  when dsb-1(5A) was first introduced and serve as evidence to rule out the contribution from DSB-2.9. In Figure 5, it is interesting that mutating a subset of conserved S/T-Q sites can rescue dsb-2 mutants, but not in pph-4.1 mutants. Although representative images of RAD-51 staining were shown in Figure S4, it will be great to compare the levels and kinetics of RAD-51 foci in these dsb-1 phosphomutant series  similarly to what's shown in Figure 4.10. In Figure 5B, in both graphs, the labels are overlaid on top of each other, it is hard to see the data point for each genotype. In the current layout of the figure, it appears that pph-4.1 data series are included in the graph on the left, although the rescue of Him phenotype was not mentioned in the text. I would suggest separating the dataset for dsb-2 and pph-4.1 and presenting separate trends with fewer data sets on each graph so that the colors and shapes of the datapoints are clearly visible.11. In Figure 5C, please indicate in the label that two lanes on the far right are loaded with 1.7x of the samples shown in the middle.Reviewer #3 (Recommendations for the authors):In several places, the writing, and order of presentation lack a bit of clarity, and some controls are missing.More specifically, several points should be addressed by the authors, listed below in order of appearance (the lack of line numbers and the dense text presentation did not facilitate the review process):– Page 1, right column, 2nd paragraph: typo: "both ATM and ATR kinases"– 2 lines below: for clarity, specify which cases you are talking about. I guess it is "in both budding yeast and mice".– Page 1 after "scaffold for the Spo11 core complex" the authors may cite also the Garcia et al. Nature 2021, which also proposed the "platform" formed by the RMM complex for Spo11 cutting.– Page 2, right column end of 1st paragraph, following paragraph and Figure 1B-1C and 1D: the explanation for why γ irradiation was used in some experiments should be provided here. It is not clear at all at this moment and comes too late in the paper.– Page 3 left column and Figures 1G, H, it would be important to show the single atm mutant – Same column and Supplemental Figure 2C: the effect of atm on rad-54 DSB levels is extremely modest and seen only at pachytene, although significant: this should be emphasized because it is an important finding. Maybe something like " "atm-1; rad-54 germlines showed…showed a level of foci slightly exceeding that of the control…".– Same paragraph, the explanation "Since homozygous mutation of atl-1..replication errors", justifying the use of heterozygous atl-1 mutants, should appear earlier, before "In contrast we found that heterozygous mutations…"S. cerevisiae. At least, this should be discussed in the Discussion.– Same page, right column, 1st paragraph and Figure 2B, C: this is not required, but have the authors tried to make phosphomimetic mutants of DSB-1 for the same residues? This should lead to constitutively low DSB numbers, as was seen for rec114 mutants in – Same paragraph: at this point, the authors should cite the study by Falk et al. (2010) that studied the meiotic phenotype of PP4 (pph3∆) mutants, even if the pleiotropic phenotype rendered the interpretation complicated. In that mutant, no reduction of DSB numbers was observed.– Page 4, left column, 1st paragraph and Figure 3A, B: why is the effect of the dsb-1 (5A) mutant alone on pairing not shown? Does it also show earlier pairing, maybe because of increased DSB numbers? This is an important control that would facilitate the interpretation.– Same page, right column 2nd paragraph and Figure 5A: the combination of mutations chosen is a bit confusing. Why was the S137-S186 combination mutant not tested, since these are the only 2 conserved positions?– Page 5 left column, last paragraph of the results: still regarding the order of presentation of the results, the fact that a smearing of GFP-DSB-1(5A) mutant is still seen should be mentioned earlier in the results, upon the first description of the 5A mutant since indeed it shows that other residues may still be phosphorylated. Does this remaining smearing in the 5A mutant depend on ATL-1 ? This needs some explanation, after reordering the results presentation.– Page 5 right column, 2nd paragraph: the part of 3D predictions using α-fold, currently in the Discussion, adds a lot to the paper and the mechanistic understanding of the respective roles of DSB1 and DSB-2. I leave it up to the authors, but it may be also moved as a paragraph to the Results section.– Same paragraph: the nomenclature should be homogenized for the trimers: either DSB-1:2:3 or DSB-1:DSB-2:DSB-3.– Page 6, last paragraph of the Discussion: as mentioned earlier, the fact that DSB was not reduced in the yeast pph3∆ should be mentioned, either here, or earlier (page 3 right column). Reviewer #1 (Recommendations for the authors):Specific points:1. What is the rationale for going straight into DSB-1? While it is a perfectly reasonable target to investigate, an explanation for the non-expert would be beneficial.This is a good point and we have added “DSB-1 is absolutely required for DSBs, whereas loss of DSB-2 still allows a low level of DSB initiation” to the beginning of the Results section (line #151), to justify our initial selection of DSB-1.2. For clarity, what is the length of the RNAi treatments? Along this same line, Figure S1B uses a combination of allele+RNAi. The choice of depletion/loss of function in the different figures should be explicitly explained.We have listed the RNAi treatment length in the methods and Results, and explained our choice to use allele+RNAi in the Results (line #157): we needed to use a balanced (mixed) population of homozygous and heterozygous mutants to obtain a large amount of material, instead of manually picking many homozygous, so used RNAi to maximally deplete PPH-4.1 protein in the heterozygotes.3. When mentioning the DSB-1 shifts in Figure S1, the authors greatly simplify these results by stating that only in the mre-11 and rad-50 mutants DSB-1 phosphorylation is absent or significantly reduced. However, htp-3 also seems to lead to significantly reduced DSB-1 phosphorylation. The authors should change these qualitative observations to a more quantitative method (maybe phospho vs non-phospho ratio?), preferably using a detection method with a decent linear range (i.e. LiCor).spo-11, chk-2, htp-3, rad-50, mre-11) also have reduced or absent DSB-1 phosphorylation; of these five, only the last two (rad-50 and mre-11) do not display an increase of DSB-1 phosphorylation upon γ-irradiation since RAD-50 and MRE-11 are required for ATM/ATR activation. We have tried to make the text clearer on this point.This is not exactly what is stated in our manuscript: the experiment the reviewer refers to here is one in which we show that five mutant backgrounds that lack DSB formation  mutants is dramatically reduced, a smearing is still present in the dsb-1(5A) mutant, indicating some other of the 86 Ser/Thr/Tyr sites are likely still phosphorylated at lower levels. We have made the text clearer now. We were not able to examine DSB-1 band shift in 1A, 2A or 3A mutants since the affinity-purified antibodies against endogenous DSB-1 were not available anymore from the Dernburg lab. We have chosen to focus the current manuscript mostly on the 5A allele since it completely lacks ATM/ATR consensus sites and is thus the most straightforward to interpret; we would like to address this issue in the future elsewhere.DSB-1 is a Ser/Thr-rich protein: possessing 91 residues of mostly Ser/Thr and some Tyr in the length of total 385 amino acids. While the phospho-DSB-1 band in 6. I got confused with the different IR doses used. For example, the result in Figure 1D suggests to me that 100 Gy would be the best to analyse the effect of kinase mutants, but in Figure 1E 10 Gy is used. Could this be clarified?We chose 10 Gy rather than 100 Gy for that experiment because 100 Gy of irradiation leads to high levels of embryonic inviability, a condition we wished to avoid to place the result more firmly in the realm of physiological relevance; also, since the result was already clear with 10 Gy, it was also the more technically convenient experiment to perform. We have indicated in the text that 10 Gy was sufficient for the experiment, and have indicated the dose used for all experiments in Figure 1D, E; Figure 1—figure supplement 1C, D and Figure 3D, E.7. Also in Figure 1E, it would be interesting to see the effect of the single atl-1 and atm-1 mutants on DSB-1 phosphorylation.atl-1 knockouts have massive DNA damage in the pre-meiotic proliferative germline due to unrepaired replication errors, leading to ATM hyperactivation. Our data (not shown in the manuscript) suggests phosphorylation of DSB-1 happens in both atm-1 single and atl-1 single null mutants; but since the genetic data shows that atl-1 and not atm-1 mutation rescues pph-4.1 mutants, combined with the fact that atl-1 AID-depletion leads to increased breaks specifically in meiotic prophase, we conclude that ATL-1 is the more physiologically-relevant kinase inactivating DSB-1 in the wild-type situation.We agree, but such experiments are complicated by the fact that 8. In Figure 5, the shifts do not seem equal. For example, the shift in the gfp::dsb-1; pph-4.1 seems significantly higher than the other ones. Have the authors tried phos-tag gels to get some insight into the possibility of different DSB-1 phospho-isoforms? At least this should be mentioned to make it clear that this is a complex analysis in spite of analysing through a 'simple' shift in a gel. In this Figure, the difference in total DSB-1 levels in the different conditions is very big, again pointing to the possibility that stability (linked to phosphorylation or not) plays a role.We agree with this observation, and think it likely that DSB-1 may be phosphorylated on non-SQ serines and/or threonines (which make up 20% of the protein). This possibility is now explicitly stated at the end of the Results section. We have tried phos-tag gels to detect GFP-DSB-1 but failed to even detect GFP-DSB-1 consistently with phos-tag gels in our hands. We have also attempted to purify tagged DSB-1 proteins from worm lysates as well as from human cell culture systems expressing worm DSB-1 to identify all the phosphorylation sites on DSB-1 by mass spec. However, the majority of tagged DSB-1 proteins end up in a detergent-resistant, insoluble fraction during the lysis and fractionation procedures, and we were not able to recover a sufficient amount of DSB-1 protein for mass spec identification of its phospho sites. We plan to address this issue in a future study.Reviewer #2 (Recommendations for the authors):My specific points are listed below:1. Throughout the paper , the status of DSB-1 phosphorylation was assessed by the band shift in western blot analyses of DSB-1. Quantification of the fast and slow migrating DSB-1 and presenting their ratio from biological replicates will strengthen this claim and show the degree to which PPH-4.1 contributes to DSB-1 dephosphorylation.Where possible we have now performed and appended quantification of phosphorylated versus unphosphorylated DSB-1 bands: in Figure 1A and Figure 5C. Since affinity purified antibodies probing untagged DSB-1 protein were not available anymore in the Dernburg lab, we were unable to quantify DSB-1 bands in Figure 1B, C, E or Figure 1—figure supplement 1C, D. However, since conclusions from these panels were based on presence/absence of an upper band and not a quantitative ratio, we do not believe the lack of quantitation affects our conclusions in those cases. The quantitation we were able to perform has placed those conclusions on a sounder footing, and we thank the reviewer for the suggestion.2. In Figure 1B, please explain in the text why irradiated worms were used for this experiment. While it was shown in Figure 1D that the level of DSB-1 phosphorylation is increased upon irradiation, the context needs to be provided when it was first shown in the paper.pph-4 RNAi treated worms.We agree that showing IP samples from γ-irradiated worms  without explaining the effect of γ irradiation was confusing. We have replaced Figure 1B with a blot image of the λ phosphatase assay using non-irradiated, 3. In Figures 1F-H, the authors tested how ATM-1 or ATL-1 kinases oppose PPH-4.1 activity by comparing the kinetics of RAD-51 loading in various single and double mutant series and showed that reducing the gene dose of atl-1, but not the atm-1 mutation, restores RAD-51 loading of pph-4.1 mutants. Have the authors examined the band shift of DSB-1 in single mutants of atm-1 or atl-1 on western blots? This will directly test whether ATL-1, but not ATM-1, is the major kinase responsible for DSB-1 phosphorylation.atl-1 knockouts have massive DNA damage in the proliferative germline due to unrepaired replication errors, leading to ATM hyperactivation. Our data (not shown in the manuscript) suggests phosphorylation of DSB-1 in both atm-1 single and atl-1 single null mutants; but since the genetic data shows that atl-1 and not atm-1 mutation rescues pph-4.1 mutants, combined with the fact that atl-1 AID-depletion leads to increased breaks specifically in meiotic prophase, we conclude that ATL-1 is the more physiologically-relevant kinase inactivating DSB-1 in the wild-type situation.This was also suggested by the reviewer #1 (specific question #7). We agree, but such experiments are complicated by the fact that 4. In Figures 1H and 2C, the kinetics of RAD-51 appearance/disappearance differ between atl-1/nT1 and pph-4.1; atl-1/nT1 and between dsb-1(5A) and pph-4.1; dsb-1(5A). The authors explained this in the text, implicating PPH-4.1 in processing recombination intermediates. Could this also (or rather) reflect the requirement of PPH-4.1 in proper synapsis and thus the delayed meiotic progression in pph-4.1 mutants? Have the authors examined SC assembly in pph-4.1; atl-1/nT1 and pph-4.1; dsb-1(5A) mutants?pph-4.1 mutants . Other studies have shown that loss of or defects in SC assembly do not impede DSB formation or RAD-51 loading , . In addition, non-homologous synapsis has never been shown to lead to delayed DSB formation in C. elegans. We therefore think it unlikely that improper synapsis in pph-4.1 mutants per se delayed RAD-51 loading. However, in the 2014 work we observed cell cycle delays in young pph-4.1 mutants, possibly deriving from hyperphosphorylation of other PPH-4.1 targets, which could account for the delays in RAD-51 appearance we see. We now mention this possibility in the main text (line #266).Previously we reported that chromosomes are fully but non-homologously synapsed in pph-4.1, pph-4.1; atl-1/nT1 and pph-4.1; dsb-1(5A) mutants by SYP-2  and HTP-3  staining . All three mutants show wild-type timing and completion/integrity of SC assembly (whether between homologous or non-homologous chromosomes). More DSBs generated either in the atl-1/nT1 or dsb-1(5A) background increased the number of homologously synapsed chromosomes in pph-4 mutants. We have added these points to the Results section (line #314) and Figure 3—figure supplement 1.To address the reviewer’s question, we have now examined SC assembly in 5. On page 4, in the final sentence of the 1st paragraph, the authors stated, "these results indicate that introduction of DSBs into pph-4.1 mutants also leads to increased fidelity of pairing and synapsis". However, the status of SC assembly was not examined in the current work. Examining the SC structure would be also important in interpreting the rescue results with the dsb-1(1A) in Figure 5, as it will further elucidate the role of PPH-4.1 in other meiotic processes.pph-4.1; dsb-1(5A) and pph-4.1; dsb-1(1A) mutants . As mentioned above, pph-4 single mutants exhibit non-homologous synapsis, but the timing of SC assembly is comparable to the wild type. Both pph-4.1; dsb-1(5A) and pph-4.1; dsb-1(1A) mutants showed normal timing of SC assembly whereas higher level of homologous synapsis is detected only in pph-4.1; dsb-1(5A) mutants compared to pph-4.1; dsb-1(1A) mutants. The pph-4.1; dsb-1(1A) mutants, which have fewer DSB formation than pph-4.1; dsb-1(5A), did not exhibit greater rescue of non-homologous synapsis. This suggests that greater levels of DSBs, found in 5A mutants, are needed to restore homologous pairing and synapsis in the pph-4 mutant background, in which SC proteins tend to polymerize in a promiscuous manner. This information has now been added to the main text , Figure 3—figure supplement 1 and Figure 5—figure supplement 2.We agree with this point. We have now examined homologous synapsis by SYP-2 , HTP-3  and ZIM-3 (Chr I and IV pairing centers) staining in 6. In Figure 3D, since the dose of irradiation is important for this experiment, please indicate that 50Gy of γ-ray was used on the graph.This change has now been made.7. When comparing the degree of rescue shown in Figure 3C and 3D, pph-4.1; dsb-1(5A) animals seem to be more proficient in chiasma formation than pph-4.1 γ-irradiated (50 Gy). How do the DSB levels compare between dsb-1(5A) and 50 Gy irradiation? Can this be explained by the level of DSBs induced?pph-4.1 and pph-4.1; dsb-1(5A) animals and found that the 50 Gy γ-irradiated animals actually have higher focus numbers, meaning that rescue of bivalents in pph-4.1 mutants cannot be simply understood as a function of how many breaks are made. The timing and nature of DNA breaks are different between γ-irradiation and 5A mutation: γ-irradiation generates excess levels of single as well as double strand breaks all at once (over the approximately 1-hour period of irradiation) whereas the 5A mutation generates DSBs gradually at normal kinetics. These differences may lead to different efficiency of DSB conversion to COs in the pph-4.1 mutant background. We now point out this difference in the Results (line #346) and discuss possibilities in the Discussion (line #551).We have qualitatively examined the levels of RAD-51 foci in γ-irradiated 8. In the current flow of the paper, I feel that Figure 4 doesn't add much insight into the overall story, as it is somewhat expected . Perhaps, this could be shown earlier  when dsb-1(5A) was first introduced and serve as evidence to rule out the contribution from DSB-2.dsb-2 mutants (panel C), the main points we convey in Figure 4 (panels A and B) are (1) DSB-2 is unnecessary for the 5A allele of dsb-1 to promote a level of DSBs that are higher than wild-type, and (2) conversely, the loss of breaks in dsb-2 mutants depends on the presence of SQ sites in DSB-1. This then sets up Figure 5 where we address the relative importance of these SQ sites in terms of their effect on dsb-2 mutants. In panel 4C, we show for completeness that the increased breaks induced by the 5A allele suffice to rescue the viability of dsb-2, in a manner similar to exogenous γ-ray-induced breaks. We would prefer to keep our figures in the present order, and have attempted to clarify these main points in Figure 4. We have added another citation of the Rosu et al. paper where this γ-ray rescue of dsb-2 was first shown. (line #375)While it is true that exogenous breaks from any source would be expected to rescue 9. In Figure 5, it is interesting that mutating a subset of conserved S/T-Q sites can rescue dsb-2 mutants, but not in pph-4.1 mutants. Although representative images of RAD-51 staining were shown in Figure S4, it will be great to compare the levels and kinetics of RAD-51 foci in these dsb-1 phosphomutant series  similarly to what's shown in Figure 4.dsb-1(1A), dsb-1(2A), dsb-1(3A) and dsb-1(5A) mutants in Figure 5—figure supplement 1. All the mutants containing the S186A conversion exhibit an increased number of DSBs compared to wild type either in early prophase (1A and 3A mutants) or throughout the meiotic prophase (5A mutant), and among these the dsb-1(5A) mutant has an extremely high number of DSBs exceeding even the wild-type level. These results suggest that a greater number of DSBs are needed to rescue pph-4.1 mutants compared to dsb-2 mutants. This difference is likely because PPH-4.1 has multiple, independent roles in homologous pairing, synapsis, DSB formation and cell cycle progression as we have shown previously  whereas DSB-2 functions exclusively for DSB formation. Higher levels of DSBs may be needed to restore homologous pairing and synapsis in pph-4.1 mutant background, in which SC proteins tend to polymerize in a promiscuous manner.We have now compared the levels and kinetics of RAD-51 foci between pph-4.1 mutants.In general, PP4 is known to have diverse substrates, and previous studies have shown that PP4 homologs in yeast and mammals are involved in resection of DSBs and loading of RAD-51 during mitotic and meiotic cell cycles , raising the possibility that PPH-4.1 may also contribute to timely processing of recombination intermediates downstream of DSB formation. Conversion of recombination intermediates to COs may be inefficient in pph-4.1; dsb-1(1A) and dsb-2;dsb-1(1A) mutants in the Results (line #408) and Discussion (line #571) sections.We now discuss possible explanations for the difference of embryonic viability between 10. In Figure 5B, in both graphs, the labels are overlaid on top of each other, it is hard to see the data point for each genotype. In the current layout of the figure, it appears that pph-4.1 data series are included in the graph on the left, although the rescue of Him phenotype was not mentioned in the text. I would suggest separating the dataset for dsb-2 and pph-4.1 and presenting separate trends with fewer data sets on each graph so that the colors and shapes of the datapoints are clearly visible.This is an excellent suggestion and we have modified Figure 5 accordingly to be visually simpler and clearer; also, since the rescue of Him phenotypes is ancillary to the rescue of viability here, we have omitted it to save space.11. In Figure 5C, please indicate in the label that two lanes on the far right are loaded with 1.7x of the samples shown in the middle.This has now been done.Reviewer #3 (Recommendations for the authors):In several places, the writing, and order of presentation lack a bit of clarity, and some controls are missing.More specifically, several points should be addressed by the authors, listed below in order of appearance (the lack of line numbers and the dense text presentation did not facilitate the review process):– Page 1, right column, 2nd paragraph: typo: "both ATM and ATR kinases"This error has now been corrected.– 2 lines below: for clarity, specify which cases you are talking about. I guess it is "in both budding yeast and mice".Yes, it is supposed to be “in both mice and budding yeast”, the statement has been clarified in the text now.– Page 1 after "scaffold for the Spo11 core complex" the authors may cite also the Garcia et al. Nature 2021, which also proposed the "platform" formed by the RMM complex for Spo11 cutting.This paper has now been cited properly as .– Page 2, right column end of 1st paragraph, following paragraph and Figure 1B-1C and 1D: the explanation for why γ irradiation was used in some experiments should be provided here. It is not clear at all at this moment and comes too late in the paper.pph-4.1 RNAi treated worms.We have now explained in the text (line #181) that we used irradiation to activate ATM and ATR kinases when we show the western blot result in Figure 1C and D. We have also replaced Figure 1B with a blot image from non-irradiated, – Page 3 left column and Figures 1G, H, it would be important to show the single atm mutant atm-1 single mutants , which is why the more sensitive rad-54 background was used in our study. In the interest of streamlining our story we chose not to pursue single atm-1 mutants in Figure 1G, H since we saw no interaction with pph-4.1.This is correct; Li and Yanowitz (2019 Genetics) have shown that RAD-51 foci are changed very little compared to WT in – Same column and Supplemental Figure 2C: the effect of atm on rad-54 DSB levels is extremely modest and seen only at pachytene, although significant: this should be emphasized because it is an important finding. Maybe something like " "atm-1; rad-54 germlines showed…showed a level of foci slightly exceeding that of the control…".atm-1;rad-54 mutant compared to control in late pachytene the text now (line #233).This is correct and we have emphasized the “slightly” increased RAD-51 foci in – Same paragraph, the explanation "Since homozygous mutation of atl-1..replication errors", justifying the use of heterozygous atl-1 mutants, should appear earlier, before "In contrast we found that heterozygous mutations…"atl-1 heterozygous mutation (line #211).This is right and we have moved this explanation up to before Figure 1G and H, where we described the effect of S. cerevisiae. At least, this should be discussed in the Discussion.– Same page, right column, 1st paragraph and Figure 2B, C: this is not required, but have the authors tried to make phosphomimetic mutants of DSB-1 for the same residues? This should lead to constitutively low DSB numbers, as was seen for rec114 mutants in dsb-1 phosphomimetic mutants in which the five serines are substituted with either aspartic acid or glutamic acid. However, both of these dsb-1 phosphomimetic mutants exhibit wild-type levels of RAD-51 foci and embryonic viability. A plausible explanation is that these phosphomimetic mutations do not functionally simulate the phosphorylation of DSB-1. We have included this information in the main text (line number #282).Yes, we have made – Same paragraph: at this point, the authors should cite the study by Falk et al. (2010) that studied the meiotic phenotype of PP4 (pph3∆) mutants, even if the pleiotropic phenotype rendered the interpretation complicated. In that mutant, no reduction of DSB numbers was observed.This has been now cited.– Page 4, left column, 1st paragraph and Figure 3A, B: why is the effect of the dsb-1 (5A) mutant alone on pairing not shown? Does it also show earlier pairing, maybe because of increased DSB numbers? This is an important control that would facilitate the interpretation.dsb-1(5A) single mutants as a control. We have now added the FISH data of dsb-1(5A) single mutant in Figure 3A and B, which shows wild-type timing of pairing.We agree it is important to show the pairing of – Same page, right column 2nd paragraph and Figure 5A: the combination of mutations chosen is a bit confusing. Why was the S137-S186 combination mutant not tested, since these are the only 2 conserved positions?dsb-1 non-phosphorylatable mutant series stepwise using CRISPR-Cas9 gene editing, and the maximum length of synthesized homology templates limited our ability to create all possible combinations. While it could be informative to test further mutations, we believe the current set suffices to support our main conclusions.We generated the – Page 5 left column, last paragraph of the results: still regarding the order of presentation of the results, the fact that a smearing of GFP-DSB-1(5A) mutant is still seen should be mentioned earlier in the results, upon the first description of the 5A mutant since indeed it shows that other residues may still be phosphorylated. Does this remaining smearing in the 5A mutant depend on ATL-1 ? This needs some explanation, after reordering the results presentation.dsb-1(5A) mutation’s effect in Figure 2 first, we prefer to keep the western blot analysis of 5A mutants compared to controls and dsb-2 mutants in Figure 5, because the blot image highlights the effect of each genotype on the relative ratio of phos/unphos DSB-1. Therefore we would like to keep the current flow of the story. Instead of changing the order of figures, we have made it clearer in the text that DSB-1 is a Ser/Thr-rich protein (non-SQ serines and/or threonines make up 20% of the protein) and it is very possible that DSB-1 could be phosphorylated at these non-SQ sites. (line #441)Thank you for the suggestion. Although we introduce the – Page 5 right column, 2nd paragraph: the part of 3D predictions using α-fold, currently in the Discussion, adds a lot to the paper and the mechanistic understanding of the respective roles of DSB1 and DSB-2. I leave it up to the authors, but it may be also moved as a paragraph to the Results section.We agree and we have moved it to the Results section now. Thank you for the suggestion.– Same paragraph: the nomenclature should be homogenized for the trimers: either DSB-1:2:3 or DSB-1:DSB-2:DSB-3.We have standardized referring to the models as (e.g.) "DSB-1:DSB-2:DSB-3".– Page 6, last paragraph of the Discussion: as mentioned earlier, the fact that DSB was not reduced in the yeast pph3∆ should be mentioned, either here, or earlier (page 3 right column).pph3∆ mutants and cite the Falk et al., 2010 paper in the first paragraph of Discussion. (line #510)Thank you for pointing out this caveat. We now state that DSB levels were not reduced in


The clinical features of urticaria have not been fully illustrated.To demonstrate clinical features of urticaria in different areas of southern and northern China.In this hospital-based multicenter study, outpatients with urticaria filled in a questionnaire during the initial visit and follow-up .P < 0.001). The incidence of pain, arthralgia, and family history in southern areas was higher than that in northern areas . The leading subtypes of specified urticaria were chronic spontaneous urticaria (81.4%) and symptomatic dermographism (35.9%). The incidence of symptomatic dermographism and cold urticaria in the southern areas was lower than that in the northern areas . Allergic diseases were the most common concomitant disorders of urticaria. More than half of the patients had to avoid certain food, such as fish-prawn-crab (30.7%) and alcohol (20%). Ebastine (41.1%) was the most commonly prescribed drug. The disease duration negatively correlated with the severity of itching and number of wheals (>50/24H) .Overall, 1,715 outpatients with urticaria with a mean age of 37.86 ± 16.08 years (range = 0.5–87 years) were recruited. The median disease duration was 1.94 ± 4.31 years (range = 0–58 years). More itching was observed in the northern areas higher than that in the southern areas (99.5 vs 94.1%, This study provides a profile of clinical characteristics of urticaria in China and filled the gap in the field of regional comparative studies on urticaria. Urticaria is a common, mast cell-driven disease, presenting symptoms like wheals, angioedema, or both. The lifetime prevalence of acute urticaria (AU) is approximately 20% . Based oUrticaria can be classified according to duration and etiology although ≥2 types of urticaria can coexist in the same patient. Different types and subtypes of urticaria are characterized by unique clinical features. Among patients with inducible urticaria in a tropical country, the most common type is symptomatic dermographism 40–73%), while solar urticaria, heat urticaria, and vibratory angioedema are rarer 3%, while. At presThis prospective multicenter study was performed from 1 January 2019 to 31 December 2019. We approached 1,800 outpatients diagnosed with urticaria, among whom 1,715 (95.3%) agreed to participate in this study. All subjects were recruited from 12 tertiary hospitals in six provinces and municipalities in mainland China, including Fujian, Sichuan, Chongqing, Hubei, and Liaoning provinces, as well as Beijing, which are distributed geographically uniform in China. This study was approved by the Institutional Review Board (IRB) Committee of Beijing Friendship Hospital, Capital Medical University, China. The inclusion criterion was as follow: patients diagnosed with urticaria with no specific physical problems that might interfere with the conduct of the interview. Also, the exclusion criteria were patients with communication inability, mental retardation, dementia, severe symptoms of acute psychosis, and severe restlessness. All participants had been selected after providing orally informed consent.As a multicenter study, dermatologists involved in this study were trained in a standardized manner, and they were performed in accordance with relevant guidelines and regulations. No matter what their race, gender, age, and education are, in our study, all participants must comply with the European Academy of Allergology and Clinical Immunology, the Global Allergy and Asthma European Network, World Allergy Organization (EAACI/GA2LEN/EDF/WAO) guidelines , or the t-test or Mann–Whitney U-test. For enumeration variables, the rate (%) within the group was reported, and the Pearson chi-square test (two test) was used for the comparison between groups. Missing data were excluded from statistical analysis. A value of p < 0.05 was considered statistically significant.All data processing and statistical analyses were performed using Statistical Package for the Social Sciences . Continuous variables were presented as the median (M) and interquartile range (Q) according to the data distribution. Normality was evaluated by the Kolmogorov–Smirnov test. Statistically significant differences between the groups were determined by the There was no specific patient or public involvement in the development of the research questions, outcome measures, study design, and recruitment/conduct of the present study. Science education in regional language helps clinicians to educate patients with urticaria. In addition, to study the participants, the key findings of the study will also be disseminated to the public through science popularization education.p < 0.05). The median disease duration was 1.94 ± 4.31 years (range = 0–58 years). Among them, 38 patients (2.2%) were pregnant and 35 patients (2%) were breastfeeding. Of the selected outpatients, 95.8% of patients were diagnosed with pruritus, which was a more common disease than arthralgia (7.5%) and fever (1.3%). The results are shown in Twelve hospitals participated in this epidemiological survey and 1,715 outpatients with urticaria were recruited, including patients with acute urticaria (198 cases) and chronic urticaria . Subjects with urticaria included 43.4% men , with a mean age of 37.86 ± 16.08 years (range = 0.5–87 years). The recruited outpatients were divided into four age groups . Except for group <20, all other groups had female predominance, and the difference was statistically significant (p < 0.001). The clinical characteristics of each type of urticaria in different regions were summarized in The leading subtype of specified urticaria was chronic spontaneous urticaria . Among them, 422 outpatients were diagnosed with chronic spontaneous urticaria alone, 437 outpatients with chronic spontaneous urticaria combined with symptomatic dermographism, and 138 outpatients with chronic spontaneous urticaria combined with angioedema. Symptomatic dermographism  and heat contact urticaria  were ranked as the second and the third common types of the disease, respectively. Additionally, three or more different subtypes of urticaria coexisted in 194 outpatients. The friction test that proved positive was seen in 61.0%  of patients with urticaria (missing in 93 cases). The survey also showed that the proportion of symptomatic dermographism and cold urticaria in the northern areas was higher than that in the southern areas . About 9% of patients (154 cases) had a family history of urticaria, and there was a big gap between the southern and the northern areas . Allergic diseases were the most common concomitant disorders of urticaria, such as allergic rhinitis (18.8%) and eczema/dermatitis (13.9%). The proportion of hypertension (5.7%) was relatively higher than diabetes (1.5%) or tumor (1.2%)  .Half of the patients with urticaria had to avoid certain food items; however, only 16.4% of them considered this approach effective. The most popular food items to be avoided by patients with urticaria were fish–prawn–crab (30.7%) and alcohol consumption (20%), followed by beef and mutton (18.6%), milk (9.5%), pepper (8.6%), egg (6.3%), nuts (1.8%), and beans (1%).p > 0.05), and the proportion of patients with acute urticaria was significantly higher than patients with chronic urticaria concerning first-generation H1-antihistamines . As a non-antihistamine compound, glycyrrhizin (34.3%) was the most widely used drug, followed by traditional Chinese medicine (9.2%) and Tripterygium glycoside (8.1%). In terms of treatment options, acrivastine and hydroxychloroquine were more commonly used in patients with a disease course longer than 1 year, which is higher than that in patients with a shorter disease course, while patients with a disease course shorter than 1 year have more prescriptions for compound glycyrrhizin and olopatadine than those patients with a longer disease course; both the differences are statistically significant (p < 0.05). Therapeutic options in this visit were summarized in The second-generation H1-antihistamines were the most commonly prescribed drugs, such as Ebastine (41.1%), levocetirizine (36.2%), and olopatadine (16%). There was no significant difference between acute and chronic urticaria in patients prescribed with second-generation H1-antihistamines .After 1 week of treatment, the proportion of severe itching dropped from 34.3 to 5.3%, and the proportion of patients with more than 50 wheals per 24 h dropped from 15.6 to 2.3%. In total, 1,715 outpatients with urticaria were examined for the first week follow-up, while 737 cases (43%) had abnormal laboratory values (with missing data in 18 cases). In total, 596 outpatients with urticaria completed follow-up once per week for a period of 1 month, and their urticaria activity score 7 (UAS7) showed a significant downward trend . The duration of the disease negatively correlated with the severity of itching and the number of wheals (>50/24H) . The prevalence of CU in men might have been underestimated previously, as adult men may not be able to visit special clinics or participate in active studies in medical centers during regular working hours . The most common type of inducible urticaria is symptomatic dermographism 35.9%). This finding is consistent with previous studies –9. An in%. This fFew population-based studies have focused on epidemiology and comorbidities of chronic urticaria (CU). According to a nationwide population-based study in Korea, allergic rhinitis, drug allergies, asthma, thyroid diseases, and cancers were common comorbidities . AnotherP < 0.001). We suspect that the dry skin caused by cold and dry weather in the northern areas is one of the major factors of itching. Second, the proportion of pain and arthralgia in northern areas is lower than that in southern areas . This is consistent with many studies (p < 0.001). Perhaps familial heritability was related to ethnic aggregation, a larger sample of data is needed to support their correlation. In addition, there are currently limited studies on north–south differences in laboratory inspection; some studies suggest that there is no significant difference in the infection rate of Helicobacter pylori in chronic urticaria cases in different regions of the north and south China , which is higher than that of the patients in the southern areas  that can be recognized by allergen-specific immune cells to elicit specific immunologic reactions, resulting in characteristic symptoms . CutaneoIn terms of treatment, second-generation histamine H1-receptor antagonists (antihistamines) remain a front-line choice and are highly recommended by dermatologists, administered either separately or in combination. Due to limitations in medication guides, doubling dosage is rarely applied in practice. Overall, urticaria responded well to treatment, especially during the first week. Both the severity of itching or number of wheals were significantly reduced after 4°weeks of treatment. Interestingly, Ebastine was the preferred antihistamine prescribed by doctors. Most previous studies have demonstrated that Ebastine is generally well-tolerated. At therapeutic doses of 10 and 20°mg once every day, it had no adverse effects on cognitive functions and psychomotor performance or on cardiovascular functions . First-gAlthough glycyrrhizin is not an approved treatment for urticaria, up to one-third of the patients with urticaria, especially those patients with a disease course shorter than one year, received glycyrrhizin, which was more common than traditional Chinese medicine and even antihistamines. The oral compound glycyrrhizin is a preparation composed of glycyrrhizin, cysteine hydrochloride, and glycine. Its primary active component is glycyrrhizic acid extracted from licorice. Glycyrrhizic acid exhibits a remarkably broad spectrum of biological and pharmacological activities, including antitumor, anti-inflammatory, antioxidant, antiviral, antimicrobial, antiulcer, anti-diabetes, hepatoprotective, cardioprotective, and neuroprotective effects . The antThis study has some limitations. First, because the participants were recruited from multiple centers, selection bias is inevitable due to a non-homogeneous population and spatial distribution. Second, routine laboratory tests, including autoantibody detection and the food allergy provocation test, were not performed for all patients due to limited conditions. Finally, the final course of the disease was not determined by long-term follow-up. Nevertheless, this study holds certain strengths. This hospital-based dermato-epidemiological study among Chinese outpatients includes a relatively large number of participants. This study presents epidemiologic features of clinical urticaria in different regions, based on diagnosis (ICD-10 codes) by dermatologists in real-world China. Although prevalence estimation is still exploratory and diagnosis of urticaria based on ICD-10 codes needs to be validated, this epidemiological study may help clinicians to educate patients with urticaria in addition to providing clues for future research endeavors.In conclusion, this study provides a profile of clinical urticaria in Chinese outpatients. CSU is the most common subtype among the participants. Allergic diseases are the most common concomitant disorders of urticaria. Fish–prawn–crab and alcohol consumption are often avoided by patients. Overall, clinical urticaria responds to treatment. Ebastine is the most frequently prescribed drug by doctors.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Urticaria Real World, 2019-P2-107-01, Beijing Friendship Hospital, Capital Medical University. Written informed consent from the participants or their legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.XW, L-JL, and L-FL performed the experiments and drafted the manuscript. X-DS performed the data analyses. Y-WS prepared the figures and tables. All authors reviewed the manuscript and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Filopodia lack any Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+ in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+ influx restored by addition of extracellular Ca2+. Intracellular Ca2+ levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing.SOCE was induced by first replacing the medium with Ca2+ increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+ increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+ altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis.Depletion of the ER Ca2+ replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+ influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell’s ability to orient itself in response to attractive or repulsive cues.Because the media were devoid of ligands that activate receptors during depletion and CaVideo AbstractThe online version contains supplementary material available at 10.1186/s12964-022-00927-y. The development and differentiation of a tissue depends on the cells’ mechanisms for determining polarity. Degradation of these mechanisms has profound implications for epithelial lining tissues, where the loss of polarity is recognized as an early sign of neoplasia. In many cell types in vitro, the dynamics of filopodia assembly and disassembly are essential for maintaining polarity. This is also true of cells in vivo. In endothelial tip cells, filopodia at the leading edge increase the efficiency of cell advancement during angiogenic sprouting . They fu2+ across the plasma membrane. It is stimulated by the release of intracellular calcium from the ER downstream of receptor ligation. The Ca2+ efflux is triggered by inositol 1,4,5 trisphosphate (IP3), which stimulates the opening of a Ca2+ channel in the ER called IP3 receptor (IP3R). The reduced Ca2+ concentration in the ER lumen activates the Ca2+ sensor, stromal interaction molecule (STIM). Membrane vesicles containing STIM associate with adenomatous polyposis coli and end-binding proteins. These plus-end, microtubule-associated proteins mediate the translocation of ER fragments to the plasma membrane [2+ into the cell. This is the Ca2+ release-activated Ca2+ (CRAC) current which is identified by its electrophysiological signature . Al reviews ). TRP chr review ).2+ influx through TRP channels. Reports on growth cones showed that filopodia extension follows an elevation of [Ca2+]i but other studies, detailing filopodia responses to spontaneous Ca2+ transients in dendrites, showed the contrary  and PTEN (Phosphatase and TENsin homolog deleted on chromosome 10). This suggested an interaction of SOCE mediators with determinants of polarity i and Ca2+ influx. The results showed that STIM and TRPC1 underwent redistribution within the cells during each phase. All the distributions of SOCE mediators differed from those of an aquaporin channel unrelated to SOCE, AQP4. AQP4, an aquaporin characteristic of respiratory airway lining cells, shared compartments with both TRPC1 and STIM1. Its trafficking pattern shared properties with both as well. TRPC1 was not confined to filopodia after Ca2+ readdition, but its translocation to the cell surface was closely related to filopodia dynamics. TRPC exocytosis appeared to depend on the influx of extracellular Ca2+ through the Orai channel.In order to clarify these relationships, we attempted to discover how filopodia were affected by SOCE mediators. The Ca2+-, Mg2+-free Hanks’ balanced salt solution .An immortalized rat cell line, 1000 W, was used. It was originally generated from rat tracheal epithelium as a model of human bronchogenic carcinoma . The celTo inhibit TRPC channels, SKF96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride) was obtained from Selleckchem . SKF96365 blocked nonselective cation channel activity with IC50s of 3 to 16 µM , 66. Pic2+]i were investigated by using cell-permeable peptides, enzyme inhibitors, and receptor agonists. 1,2-dioctanoylglycerol (DOG) was obtained from Sigma-Aldrich, and the aldehyde inhibitor of calpains, ALLN, from Focus Biomolecules . Calcium-like peptide 2 (CALP2) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) were obtained from Tocris . CaM kinase II and calcineurin inhibitors, autocamtide-2-related inhibitory peptide and calcineurin autoinhibitory peptide were obtained from EMD Millipore, Temecula, CA. Myosin light chain kinase (MLCK) inhibitors, ML-7 and MLCK peptide, were obtained from Cayman Chemicals . When epidermal growth factor (EGF) was used to supplement Ca2+-free HBSS, the human recombinant protein  was used at a final concentration of 10 ng/ml.Possible mediators of the response to altered i and transient filopodia formation” section). We used 30 min as the standard interval for depleting the ER of Ca2+ except in experiments where we were testing for accelerated depletion. After Ca2+ readdition, filopodia increased for 20 min (data not shown), but their prevalence initially showed high variability. As the recovery of ER Ca2+ depended on washing out CPA, a single time was set for collecting cells. This minimized variability after Ca2+addition .Filopodia only increased if the CPA treatment time was extended to 30 min  for 20 min in culture medium, then transferring the cells to Ca2+-free HBSS with or without CPA. Calcium orange was made up at 0.25 µg/µl in dimethylsulfoxide and used at a final concentration of 2 µg/ml. Images were acquired at 5-min intervals, using the software, instrumentation and settings described below (see “Equipment and settings” section) and processed by background subtraction. The averaged intensity value per cell was calculated for each frame processed.Intracellular calcium levels were compared in cells incubated in CaFor immunofluorescence staining, cells were fixed as above and permeabilized with 50 µg/ml digitonin  and 0.2% Triton-X 100 in cytoskeletal buffer. Phospholipase C isoform  and antibodies against Orai1 and Orai3  were made in rabbit. Mouse monoclonal antibodies included anti-β-tubulin and rabbit polyclonal anti-TRPC1 from Sigma-Aldrich and antibodies against Vamp2 , CaV1.2 , caveolin-2 , β1 integrin (BD Biosciences Pharmingen), STIM1/CRACR2A , and a combination of five clones against PLCγ1 (Sigma-Aldrich).In experiments with sheep antibody against the extracellular loop of TRPC1 , primary staining was followed by Cyanine 3-labeled donkey anti-sheep antibody from Jackson ImmunoResearch . Where a rabbit and mouse primary were used, secondary staining was performed using donkey tetra-rhodamine isothiocyanate (TRITC)-labeled anti-mouse IgG and goat fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibodies (Jackson ImmunoResearch). Triple staining procedures were done using the secondary antibodies, donkey anti-mouse  tagged with Cyanine5, Cyanine 3-tagged anti-sheep, and FITC-tagged anti-rabbit. Samples were mounted in 2.5% DABCO made up in 2,2′-thiodiethanol (Sigma-Aldrich) and viewed with a 100 × lens as described below (see “Equipment and settings” section). Control samples that were unexposed or exposed to irrelevant antibodies, followed by the secondary antibodies, showed no staining.2+ concentration, we used a confocal Leica DMI3000B inverted microscope  equipped with a Lumen Dynamics X-Cite light engine and Leica rhodamine filter set  and 10 × lens. Images were acquired once per minute with a Rolera Thunder cooled CCD camera with back-thinned, back-illuminated, electron-multiplying sensor . MetaMorph version 7.8, 4.0,  was used to configure the hardware settings for time-lapse recording.To assess intracellular CaSamples triple-stained for the SOCE mediators, STIM1, Orai1/3, and TRPC1, were also imaged with the Leica DMI3000B inverted microscope using Spectra X LED source  and X-Light spinning-disk confocal unit  with Semrock bandpass filter FF01-440/521/607/700–25 and dichroic FF410/504/582/669-Di0l-25 × 36. The software and CCD camera used were those identified above.For studies by higher magnification, epifluorescence images were taken on an Axiophot microscope  using Zeiss 100 × Plan-Neofluar/1.30 objective lens and FluoArc mercury lamp. Filter sets for FITC  and Cyanine3 or TRITC  were from Chroma . Images were acquired with an Andor camera  running under Molecular Devices MetaVue software. The final pixel size was 63 nm.To determine the role of water entry in filopodia formation, bovine serum albumin  was made up in Waymouth’s medium  at 33.35% (w/v) and diluted 3:4 with medium before use. Cells were cultured on germanium coverslips and maintained at 37 °C while being viewed. Time-lapse recordings were made using a Plan-apochromatic 100 × lens in the Zeiss Axiophot microscope, with image acquisition as described above (see “Equipment and settings” section).Pearson correlation coefficients were obtained for matched images of thin, peripheral areas viewed in the FITC and tetra-rhodamine channels. The Colocalization Finder  plugin, The different loci, represented by immunocytochemical localizations of their intracellular sites, were segmented from images of thin, peripheral portions of the cells. The selection of these areas obviated the complication of multiple layers of structures, as overlapping layers of particles were absent. Contrast enhancement and edge detection operations were run on each image in ImageJ. Then, the threshold was adjusted to maximize the number of continuous outlines around figures. The figures’ sizes were measured in pixels, using the particle analysis plugin and a circularity setting of 0.9–1.0. The radius was calculated by modeling the number of pixels in each locus as a circle, and the relative size distributions were compared on plots. As the images used to generate the data were partial images of cells, and some cells lacked areas suitable for particle analysis, the number of images subjected to analysis was used as the sample number in statistical tests.To determine the diameter of the zone occupied by TRPC1 clusters at the cell surface, we estimated the maximum extent of the zone around each filopodium using the line function of ImageJ. The analysis was conducted around the zone of each filopodium where the loci were clearly in-focus. Loci, referred to as particles here, were assigned to the maximum distance, and the results were plotted as a cumulative distribution of distances.2+ in the presence of CPA. It is well-established that this depletes the ER of Ca2+ and causes the formation of a STIM1-Orai complex at the plasma membrane. Ca2+ deprivation increased the filopodia briefly. This was followed by a spontaneous decline to the starting level by 30 min. In a second step, refilling of the ER is initiated by CPA washout and replacement of the Ca2+-free medium with Ca2+-replete medium. After Ca2+ readdition, the prevalence of filopodia rose . To determine the effect of CPA in Ca2+-replete solution, we conducted the experiment with Ca2+ present at all stages. This had a negligible effect on the number of cells with filopodia  pump by CPA, which prevented Ca2+ being returned to the ER. After 15 min of CPA treatment, [Ca2+]i began to increase, while filopodia declined .When CPA was washed out and extracellular Caain Fig. d. This wr review ). Duringcf. Fig. a, b, d. cf. Fig. d, f. How2+ influx” section). The morphology of the filopodia was known from previous studies i was either low and rising or high and falling i within 1–3 min. Then, the level spontaneously declines to baseline [2+]i increases and decreases are slower . After CPA washout and Ca2+ readdition, global [Ca2+]i was falling despite the obvious display and activation of TRPC channels. If cell depolarization was induced simultaneously with Ca2+ readdition, a rapid rise of Ca2+ could be observed due to VACC activation. VACCs reside within laterally confined regions of the plasma membrane, and their activation is expected to elevate local Ca2+ to micromolar concentrations. Indeed, at these higher levels, TRP channels disseminated on the cell surface may be closed by CDI . Of these two channel classes, the VACCs are more highly selective for Ca2+ . Na+, w2+ addition. We propose that the choreographed movements of TRPC vesicles after Ca2+ readdition are explained by the function of Orai channels, which is supported by previous evidence that Orai works in cooperation with TRPC-bearing vesicles i, Ca2+ binding to PLC may cause a conformational change and expose sites for docking molecules activated by signaling from G protein-coupled receptors and tyrosine kinase receptors (see for review [2+ directly (see below) but may be activated by binding a Rho family GTPase. Secondly, PIP2 serves to nucleate actin filaments by activating profilactin [2+ influx enabled filopodia extension.Some concentration of PIP2 was essential to activate TRPC channels , but itspossible . Neverthpossible . In memburvature . This gar review ). As menfilactin  and by sfilactin , (see fofilactin ). In mosfilactin , 37. ThuDrosophila signalplex, which organizes a number of signaling proteins on a scaffold protein containing multiple PDZ motifs, called INAD . INAD is attached to the plasma membrane by TRP/TRPL, the only transmembrane proteins in the complex. Drosophila orthologues of PKC and PLCβ are recruited to additional PDZ motifs of INAD. The structure of the putative mammalian signalplex is similar, although assembled on NHERF which has fewer PDZ motifs. During ER depletion, TRPC1 clusters resembled a sort of signalplex, suggesting that channels in this form were inactive. Nevertheless, the colocalization shows that they were in close proximity to PLC. Because clusters were dispersed after Ca2+ readdition, TRPC was simultaneously activated and detached from the scaffold. This is consistent with previous reports that PIP2 enhanced the formation of the mammalian TRPC1/4 and TRPC1/5 complexes P2 breakdown was maximized, it was rapidly depleted, which terminated TRP channel activation and Ca2+ influx. This was attributed to the absence of Ca2+-dependent inhibition of PLC by Ca2+ influx through TRP channels [As suggested above , a useful model for TRPC activation may be the r review ), but inr review . PIr review . Despitechannels , (see fochannels ).3, is itself an activator of TRPC channels through SOCE. Because the current experiments show that a long time elapsed between SOCE induction and the stimulus for filopodia extension, these signaling pathways are not considered influential in extension. The turnover of PIP2 appears to be regulated by opposing effects of Ca2+ on PLCβ. Whereas it is not clear that the PLCβ isoforms are Ca2-activated, Ca2+ stimulates the in vitro catalytic activity of PLCδ and η over 2–3 orders of magnitude [2+ but rather cause a conformational change that may activate the GTPase-activating activity of PLC, thereby releasing the bound GTPase . A simil effect . As the 2+ readdition stimulates TRPC secretion” section), dissemination of TRPC1 throughout the cell surface was closely related to filopodia extension. Channel gating, by whatever means (see “Role of PLC in TRPC activation” section), does not explain their dissemination. It should be noted that, in the putative mammalian signalplex, NHERF is recruited to TRPC4/5 and in turn recruits ERM . Moreover, the PLCβ isoenzymes bind to NHERF and turn over PIP2. As ERM proteins are ubiquitous organizers of the subcortical actin filaments, the signalplex may be supported by an actin network. ERM proteins are also bound to the plasma membrane via PIP2 (see for review [As noted above (see “Car review ) and arer review . If ERM r review , 70, we 2+ replenishment” section). This was consistent with previous evidence implicating Rab4-dependent recycling in the exchange of TRPC vesicles between intracellular compartments and the plasma membrane [2+ influx likely occurred over the entire cell surface under the current experimental conditions. However, under conditions of receptor-ligand interaction, signaling would have directional significance and thus, both STIM migration and TRPC1 exocytosis may have a directional bias.As mentioned above, vesicles from the RRP fuse with the plasma membrane depositing additional TRPC1 channels on the cell surface P2 renewal by “PIP2 treadmilling” and assures a steady supply of the molecules required for filopodia extension. Some proteins that participate in filopodia assembly, for example the WAVE complex, Enabled, and IRSp53, were bound to PIP2 P3 [The life cycle of the filopodium comprises four phases, namely initiation, extension, persistence, and retraction. The evidence suggested that extension was the phase regulated by Ca3,4,5)P3 . If wort3,4,5)P3 , 55, 111Both ERM dissociation from the plasma membrane and exocytosis from the RRP might be able to disrupt the subcortical network, so that either may create sites for rearranging the existing filaments or assembling new filaments. It has been proposed that there is a protein that “seeds” patches of membrane for filopodia extension (see for review ). AlthouAdditional file 1: Supplementary Figures and Tables.