Our results demonstrate the efficacy of fourSig with previously published and novel 4C-Seq data sets and show that our significance prioritization correlates with the ability to reproducibly detect interactions among replicates.The ability to correlate chromosome conformation and gene expression gives a great deal of information regarding the strategies used by a cell to properly regulate gene activity. 4C-Seq is a relatively new and increasingly popular technology where the set of genomic interactions generated by a single point in the genome can be determined. 4C-Seq experiments generate large, complicated data sets and it is imperative that signal is properly distinguished from noise. Currently, there are a limited number of methods for analyzing 4C-Seq data. Here, we present a new method,  The organization of chromosomes in 3D space has a significant impact on mammalian gene expression . VariatiCircular chromosome conformation capture with sequencing (4C-Seq) is a variant of 3C technology that identifies the range of genomic interactions formed by a specific locus (e.g. a gene promoter) ,13,14. CfourSig, a conceptually simple yet powerful statistical method for analyzing 4C-Seq data. fourSig can analyze data from a wide variety of 4C-Seq protocols, uses the full range of quantitative information captured by 4C-Seq, provides variable parameters to adjust for desired resolution and offers a method of peak prioritization to categorize statistically significant genomic interactions.The currently available 4C-Seq analysis methods vary in the treatment of raw data and strategy for determining signal from noise. Further, none provide a methodology for predicting interactions that are likely to be reproduced among experimental replicates. To this end, we have developed a suite of software called fourSig is as accurate and precise as the most sophisticated existing methods, but also prioritizes the statistically significant interactions. To demonstrate further the high resolution and effectiveness of fourSig’s prioritization feature, we generated and analyzed a novel allele-specific 4C-Seq data set. We hypothesized that if long-range interactions are a general property of transcriptional regulation, 4C-Seq performed at the transcription start site (TSS) of a gene that demonstrates an allelic expression bias may reveal allele-specific interaction profiles. Such an analysis would shed further light on the relationship between genomic interactions and gene expression that is independent of sequence variation.Using previously generated 4C-Seq data sets, we demonstrate that Ibtk encodes a tyrosine kinase inhibitor that displays strong allelic expression bias in F1 hybrid trophoblast stem (TS) cells. After using fourSig to analyze allele-specific 4C-Seq data from the Ibtk locus, we validated our method by performing fluorescent in situ hybridization (FISH). While both Ibtk alleles have similar interaction profiles, we identified several interactions unique to each allele using the high-resolution analysis offered by fourSig. Interestingly, one allele-specific interaction formed predominately by the active Ibtk allele overlaps with a putative enhancer element. Taken together, our data demonstrate the utility, accuracy and precision of fourSig.fourSig is a software suite, written in Perl and R, used to identify statistically significant interactions from 4C-Seq data. 4C-Seq reads are initially mapped to the genome. Mapped data are then transformed into a database of genomic fragments using samToRetab.pl or bowtieToRetab.pl . Each database is specific to the design of the 4C experiment. This database of genomic fragments corresponds to the restriction site locations of the enzyme used to generate the 3C library (the template for the 4C library). The mappability for any given 3C fragment is then determined based on several factors: the location of 4C restriction sites (both 4C and linearization), the minimum and maximum size selected for the sequencing library, the length of potentially captured fragments, the length of the primer used for amplification and the length of the sequencing reads. After the template database is produced, the mapped reads from the 4C-Seq data are assigned to generate a file that serves as the input file for the fourSig.R program. By default, fourSig.R removes fragments marked as unmappable within the input database files. Any type of 4C-Seq data can be transformed into fourSig format, and a detailed description of the data structure may be found in the Supplementary Methods.fourSig.R allows the user to define portions of the cis-chromosome to be masked out before determining significance thresholds. The region close to the viewpoint will contain a high concentration of reads per fragment and will typically require a higher significance threshold than more distal regions. Therefore, this feature may be used to focus the significance testing to viewpoint-proximal regions. Alternatively, regions located near the viewpoint can be masked out to allow for a lower threshold for significance for regions further away. Additionally, a combination of masked analyses could be used to approximate varying thresholds in cis.fourSig.R then calculates the significance threshold, the minimum number of reads within a window required to achieve a false discovery rate (FDR) that is defined by the user. The shuffling and threshold determination are repeated as many times as desired; however, we recommend at least 1000 permutations. Finally, the user can specify the percentile from which the smallest calculated threshold that occurred (we recommend the top fifth percentile) is used as the final cutoff for significance for that chromosome. Contiguous windows that are significantly enriched with reads are then merged into a single interaction, and 3C fragments on the 5′ and 3′ ends of each domain that do not contain reads are removed. The analyses described here calculated a significance threshold from the top fifth percentile of 1000 calculations of FDR < 0.01. Parameters for background masking and window sizes varied depending on the analysis and are described in the text below.The significance testing itself is described in fourSig to prioritize interactions based on the likelihood that they are reproducible. This method is applied to the windows that comprise an interaction, and the entire interaction is categorized based on the broadest assigned designation. As explained in Additionally, we used TS cells were cultured as previously described ,18. TS cIbtk verification may be found in the Supplementary Methods.RNA was harvested from TS cells using the TRIzol reagent and the manufacturer’s procedures (Ambion), treated with DNase I and purified using the protocols described for the RNeasy Mini Kit (Qiagen). RNA preps were quantitated using a Nanodrop 8000 (ThermoFisher), and cDNA samples were prepared from 250 ng RNA using SuperScript III Reverse Transcriptase (Invitrogen). Quantitative polymerase chain reaction (qPCR) reactions were performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad). Details of the 2 plates of TS cells grown independent of feeder cells for preparation of 4C libraries were treated with 0.25% trypsin to form single-cell suspensions. Plates were pooled for fixation with 1% formaldehyde, counted by hemocytometer and stored at −80°C in aliquots of 3 × 107 cells.Batches of 10-cmGapdh locus . Test amplification of 3C libraries, no-ligase controls and bacterial artificial chromosome (BAC) ligation controls was performed using the SsoFast EvaGreen Supermix (Bio-Rad), and digestion of amplified product by HindIII.The protocol for generating initial 3C libraries and subsequent 4C libraries was adapted from previously reported studies and was modified for optimal use in TS cells ,12. 3C lIbtk were produced by digesting purified 3C products with NlaIII (NEB), performing a second proximity ligation, linearizing by digestion with NsiI (NEB). Similar to the 3C libraries, efficient digestion was tested by electrophoretic migration in 0.8% agarose. The Ibtk 4C libraries were amplified with Pfx polymerase (Invitrogen) using a double-amplification strategy with primers designed to capture an informative single-nucleotide polymorphism (SNP) on the NlaIII side of this digestion scheme. The primers for initial amplification are homologous to both CAST and B6 sequence and contain a portion of the sequencing adapters  with four random bases separating the adapter from the genomic sequence (Supplementary Table S1). Absence of amplification was observed in source 3C libraries and the 4C no-ligase controls to ensure faithful amplification of the intended target. All amplifications were performed within a linear range, as determined by an initial test with qPCR.4C library templates for Ibtk 4C intermediates were purified and size-selected (200–650 bp) by gel extraction. Size-selected intermediates were then amplified with Phusion polymerase (NEB) using primers containing sequence homologous to the portion of the sequencing adapter used in the first round of amplification and the remaining sequence needed to complete the sequencing adapters (Supplementary Table S1). Final products were purified using Ampure XP beads (Beckman Coulter) and quantitated using the Qubit Assay Kit for dsDNA before submission for sequencing. 4C libraries were submitted to the UNC High-Throughput Sequencing Facility for paired end sequencing using 2 × 150 bp on the Illumina MiSeq platform (Rep 1) and 2 × 100 bp on the Illumina HiSeq2000 platform (Reps 2 and 3).After the initial amplification, A stepwise detail of our 4C protocols can be made available on request.Mus musculus, and a genome sequence for CAST/Eij (CAST) was generated by editing appropriate nucleotides with SNPs for Mus musculus castaneus (Supplementary Methods.The C57BL/6 (B6) genome sequence was taken from the mm9 genome assembly for astaneus . Custom astaneus . Furthercis. The proportion of Chromosome 9 interactions identified as occurring in trans  was used to determine that the likelihood that this assumption leads to erroneous results is negligible (see Results).After significant interactions are assigned, allelic calls are made based on whether the number of SNPs in SNP-containing reads detected in the interaction is significantly higher than what would be expected by random chance. Chromosome 9 interactions lacking sufficient SNP coverage to make an allelic call were assumed to occur in R  were used to probe DNA at the sites of predicted interactions and the region immediately upstream of the Ibtk gene (Supplementary Table S2). Typically, the Ibtk cDNA probe was labeled with Cy3-dCTP , the fosmid for the Ibtk TSS probe was labeled with Alexa Fluor 488-dUTP (Molecular Probes) and fosmids targeting predicted interactions were labeled with Cy5-dCTP .Probes were fluorescently labeled using the BioPrime DNA Labeling System (Invitrogen). Supplementary Methods. Z-stack images were taken in grayscale at 63× using a Zeiss AxioImager M2 equipped with an AxioCam MRm camera (Carl Zeiss). Merged Z-stacks were deconvolved using the iterative algorithm in the AxioVision software package (Carl Zeiss). Distances were measured in three dimensions from the approximate centers of DNA-FISH signals using the ZEN 2011 black edition software package (Carl Zeiss). Recorded measurements were used to produce cumulative distance plots with R. To produce representative images, z-projections for each color channel were produced and exported with AxioVision, then merged and pseudo-colored with Adobe Photoshop CS5.1 (Adobe Systems).Details of the FISH procedures may be found in the Supplementary Figure S2). The end product from a 3C preparation is trimmed by restriction digestion, self-ligated and linearized. Although these processing steps generate many molecules, a certain proportion will consist of viewpoint sequence flanking an unknown sequence that was ‘captured’ due to a genomic interaction at the time of fixation.4C libraries are generated from downstream processing of 3C templates and are used to identify genome-wide interactions with a fixed locus, referred to as the viewpoint  separating them ,23. ThisfourSig uses a variety of information, including the size of the 4C primers; locations of the 3C, 4C and linearization enzymes; and the size range of sequenced molecules, to determine which 3C fragments can be identified by 4C. Fragments that are too large for the sequencer, too small to be aligned or would not have been amplified by PCR are flagged such that they can be eliminated from the analysis (see Supplementary Methods). This allows fourSig to model accurately the random distributions used for threshold calculation (described below).Before statistical analysis, W, can be altered depending on the desired resolution of the contact map. Smaller window sizes provide higher resolution results, which are useful for investigating the details of specific interactions, while larger window sizes are better for showing general trends and identifying highly reproducible interactions (described later).The schematic in fourSig allows for the regions surrounding the viewpoint to be treated separately (see below). The total number of shuffled reads per window is then tallied . Using the example demonstrated in Xs from shuffled reads were >10, so it is likely that a cutoff as low as 10 reads in a window would be an underestimate of the true threshold for FDR < 0.01. Similarly, 50% of the shuffles-generated Xs were >30, so it is likely that 30 would also be an underestimate. However, only 5% of the shuffles generated cutoffs >40, so we can be fairly confident that this threshold does not underestimate an FDR < 0.01.For each chromosome, the total number of reads in each window is tallied to generate an observed distribution B, Step 1 tallied B, Step 2ired FDR B, Step 3fourSig method was heavily influenced by a previously described 4C-Seq analysis method  resulted in significance thresholds of either 47 or 48 reads per window. While the average threshold was still 47.27, the variance was reduced by almost 10-fold to 0.22-fold . One possible explanation for the increased variance in the previous method is that the FDR calculation is dependent on averages, which can be highly influenced by extreme outliers. In practical terms, this increased variance may lead to skewed threshold calculations when the method is applied only once to a 4C-Seq data set. In contrast, the FDR calculations in fourSig are made after each randomization, and a single outlier cannot have a disproportionate effect on the other calculations. Based on this difference, fourSig should be more robust to outliers generated during read randomization, thus reducing the potential for misestimating a suitable significance threshold.The s method . Howeverdata set  and the   locus contains several well-characterized long-range interactions that associate with gene expression states  cells, where beta-globin genes are active, and fetal brain (FB) cells, where the beta-globin genes are silent. One high-resolution 4C-Seq analysis method, r3CSeq . Using equivalent settings (window size of 1 and only surrounding the Hbb1-b1 viewpoint), fourSig produced results that were nearly identical to those obtained with r3Cseq . Specifically, both methods detect interactions upstream of the viewpoint and at the beta-globin locus control region (LCR) in FL tissue when compared with FB tissue. Additionally, fourSig results are consistent with those obtained from 4Cseqpipe . Comparatively, there were approximately three times as many Broad interactions in FL cells than were observed in the FB tissue . As will be shown later, these results suggest a higher probability of reproducing the interactions near the viewpoint in the FL cells than in FB, which is consistent with LCR interactions facilitating expression of Hbb-b1 in the FL. Furthermore, these results are consistent with previous claims that larger window sizes improve reproducibility  cells  required for ES cell self-renewal, was found to be engaged in interactions with Rybp, Tcf3, Ezh2 and Smarcad1, all genes implicated in the maintenance of ES cell pluripotency. Additionally, their results showed strong signals at the Bcat1 and Kras loci. The statistical analysis by de Wit et al. (2013) was performed using a window size of 100 3C fragments; however, a much smaller window (W = 31) was used for visualization in their paper. Because the incorporation of read data in fourSig allows for relatively small window sizes, we performed an analysis using W = 31. When masking out a 50-kb region on both sides of the Nanog promoter, we also identified interactions with the Rybp, Tcf3, Smarcad1, Bcat1 and Kras loci. In addition, these loci were assigned Broad priority classifications, indicating that their significance was not derived from a single fragment within a window . Interestingly, we were unable to detect significant interactions between Nanog and the Rybp, Tcf3, Bcat1 and Kras loci with the r3Cseq software (data not shown).In addition to S) cells . The oriEzh2 was relatively small compared with the other peaks , so we used fourSig to perform a localized analysis, similar to the approach used by de Wit et al. (2013). To approximate the ∼3000 HindIII fragment background model used in the previous study, we generated threshold values from a local distribution derived from the observed data within 4.5 Mb of either side of the Ezh2 locus. This localized analysis identified a Broad significant interaction between Nanog and Ezh2 .The peak at Nanog 4C data set to characterize the effects of different window sizes on the identification and categorization of interactions by fourSig. Using window sizes of 31, 10 and 5 HindIII fragments, we identified an inverse correlation between window size and number of detected interactions . However, the proportion of Narrow categorizations as a percentage of total interactions also increases as the window size is reduced . Therefore, while a reduction in window size may lead to a greater number of called interactions, the prioritization scheme is effective at illuminating which interactions are representative of single-fragment interactions.Finally, we used the fourSig, we designed and performed an allele-specific 4C-Seq experiment at the TSS of a gene that exhibits a strong allelic expression bias.Long-range contacts have frequently been reported to have functional association to expression states ,31,32. TSupplementary Figure S2). Ultimately, we settled on Ibtk, a gene encoding an inhibitor of Bruton’s tyrosine kinase .Our laboratory previously produced allele-specific RNA-Seq data in TS cells derived from reciprocal F1 crosses between B6 mice and CAST mice . We seare kinase ,36, as aT allele A. These Ibtk, we designed a 4C-Seq strategy to identify allele-specific long-range interaction patterns . Three sequencing data sets representing two biological replicates were obtained and analyzed by fourSig. Each read is a hybrid of known and unknown sequences. The known sequence was analyzed for the presence of the B6 or CAST SNP to determine the allelic origin of the viewpoint . Additionally, the unknown sequence was also analyzed for the presence of a known SNP to determine the allele to which the interaction occurred. Before analysis with fourSig, mapped reads were sorted into separate data sets depending on the allelic origin of the viewpoint and the allelic origin of the captured sequence.Having confirmed the allelic expression bias for fourSig uses read counts to determine significance threshold, it was imperative for us to rule out any interaction called due to technical errors such as PCR amplification bias. In our assay, this distinction can be made due to the inclusion of random barcodes incorporated between sequencing adapter sequences and 4C genomic primer sequences . Repeated amplification of single contacts will generate reads containing identical barcodes on both ends of the read, creating an indicator of the degree to which amplification bias may be affecting the detection of a mapped 3C fragment. We inspected the barcodes in each of our data sets after sorting the mapped reads by allelic origin and found no fragments containing a disproportionate number of reads with identical barcodes (data not shown).Because Ibtk 4C-Seq data with fourSig using a window size of five 3C fragments and an FDR < 0.01. Using informative SNPs in the contacted regions, interactions were assigned to specific alleles (see Supplementary Methods). For the present analysis, we excluded trans-interactions mapping to the opposing Chromosome 9 of the analyzed viewpoint . Interactions mapping to Chromosome 9 that lacked informative SNPs were assumed to be in cis. Extrapolation of the average proportion of trans calls in each library would suggest that <1% of the interactions may be erroneously called cis, making this a reasonably safe assumption.We hypothesized that allele-specific contacts may harbor putative distal regulatory elements. Therefore, window sizes were kept small to achieve high-resolution results. We analyzed cis-interactions were compared among the three replicates for each viewpoint allele. The intersection of these libraries is defined as the set of consistent interacting fragments from interactions detected for each viewpoint . Because the majority of allele-specific contacts were found within 1–3 Mb of the TSS, interactions for verification were chosen from loci within this space. To ensure that the FISH test was reflective of the high resolution and precision with which fourSig calls interactions, loci for validation of allele-specific contacts were selected so that the called interactions were not isolated by large linear distances from interactions found on the opposing allele. Because SNP differences cannot be used to discriminate between alleles in FISH experiments, we used the allelic bias in Ibtk expression to identify the B6 or CAST allele. As such, the colocalization of Ibtk expression by RNA-FISH with a DNA-FISH probe for the Ibtk TSS was used to identify the active (CAST) allele . We observed monoallelic expression in 92.8% of scored nuclei, confirming that the use of RNA-FISH is an acceptable indicator for allelic discrimination (data not shown). For each experiment, DNA-FISH probes for the Ibtk TSS and the assayed interaction were cohybridized with the Ibtk cDNA probe. Measurements were taken from the centers of DNA signals . The repressed allele was consistently closer in space to the B6-specific interaction, whereas the opposite was found for the CAST-specific interaction . Of the fragments from interactions found in all replicates, none were detected by r3Cseq that were not also found in the fourSig analysis . Additionally, although the small sample size skews the exact relative proportions, we did find approximately similar proportions of overlaps between allelic interactions as determined by r3Cseq  .To determine whether the application of g r3Cseq  with defNanog analysis, r3Cseq detected consistent interactions only proximal to the viewpoint for both alleles . Although no interactions were detected further than 1.1 Mb from the viewpoint, the trend of greater dissimilarity in allelic interactions further from the viewpoint seen earlier was preserved . Notably, all of the interactions detected by fourSig and validated by FISH were beyond the range of the most distal interactions found by r3Cseq. Finally, the r3Cseq method failed to consistently detect contact with putative enhancer described in Supplementary Figure S10B). Therefore, this comparison demonstrates that despite a substantially simpler method of detecting significant interactions, fourSig is more sensitive to the detection of validated interactions occurring at further distances from the viewpoint and highly probable allelic interactions with regulatory elements that have published precedent for functional activity.An inspection of the positioning of consistent interactions from each allele found that, similar to what was observed in the fourSig, for analyzing 4C-Seq data. fourSig uses the full range of quantitative information derived from read depth, produces consistent results and can inform as to the reproducibility of interactions among replicate experiments.In this study, we introduced a new method, r3Cseq , it is possible to perform all of the permutations first and use the average of the cumulative data set to identify a single cutoff for the FDR. Initially, we used both methods, but found that the former method provides less variation among repeated analysis of data sets and is likely to be more robust to extreme values that occurred in rare permutations. Additionally, while the use of a single threshold for an entire chromosome may result in overly conservative significance calls, especially for interactions with loci at large distances, the use of the masking feature, as demonstrated for the Nanog to Ezh2 interaction, is very effective for making refined assessments at any particular region of interest. This is a powerful feature in fourSig, which can allow the user to fine tune the sensitivity of interaction detection by tailor-suiting a background model to any location or locus size desired. Taken together, fourSig provides an easy-to-use method for high-resolution detection of genomic interactions while offering the user the flexibility to find optimal conditions for gaining informative results.There are two ways to determine the threshold for significance when using permuted data. Ibtk data lend strength to this feature’s role in flagging potentially interesting interactions.The interaction prioritization feature represents a novel addition to 4C-Seq analysis and a useful improvement over existing methods. In addition to the consistent application of a Broad designation to many well-described interactions from published data sets, the trends of increased reproducibility for Broad interactions demonstrated in replicate analysis of the fourSig offers predictive value in terms of the likelihood that detected interactions may be reproduced, it should be noted that interactions classified in all three categories are capable of being consistently reproduced. Our analysis of the intersection of replicates showed that >5% of the consistently detected 3C fragments were found within interactions designated as Narrow or Intermediate. This could occur, for example, in the event that the dominant 3C fragment in a window is large, leaving little linear distance to either side of the primary point of contact to capture neighboring fragments. However, this is likely to be an infrequent event when using a 6- or 4-cutter for generating the initial 3C library. Additionally, it is not our intention to suggest that the use of this feature may serve as a replacement for performing experimental replicates. As is evidenced by the analysis of the Ibtk data, a sizable portion of the Broad interactions are not identified in all of the replicates. Instead, a more appropriate use of this feature would be to serve as a guide for emphasizing interactions in the event that few replicates are possible or as a support to avoid seeking diminishing returns yielded from excessive replicate experiments.However, while the interaction prioritization feature in Ibtk do not prove that functional regulatory elements lie within the described interactions, the consistency at which we detected allele-specific interactions supports the notion that functional association of conformation with differential expression state is a possible source of regulatory control.One biological context in which the functional role of higher-order chromatin organization may have particular importance is in allelic expression bias. Differential expression patterns between alleles are traditionally associated with genomic imprinting ,41; howeIbtk locus indicate that the majority of interactions detected within 1 Mb of the viewpoint are shared between alleles. This is consistent with numerous 4C studies that report substantially increased contacts with loci at shorter linear distances from the viewpoint  . Funding for open access charge: NIH.Conflict of interest statement. None declared.


The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles (‘bulk’ endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity.DOI:http://dx.doi.org/10.7554/eLife.01621.001 Neurons propagate electrical signals from one cell to the next using small molecules called neurotransmitters. These molecules are held inside small compartments called synaptic vesicles. Once a neuron receives an electrical stimulus, the membranes that enclose the synaptic vesicles fuse with the plasma membrane that encloses the neuron. This releases the neurotransmitters, which then trigger an electrical signal in the neighboring cell. Once the neurotransmitters are released, the vesicle membrane is rapidly reinternalized from the plasma membrane in a process called endocytosis and then recycled, ready for the next round of signal transmission.The process of synaptic vesicle membrane endocytosis and recycling has been studied extensively, and several different mechanisms by which it occurs have been identified. The best understood relies on a protein called clathrin, and is thought to be essential for nervous system function. Recently, however, a mechanism of vesicle membrane endocytosis that does not involve clathrin was identified. This mechanism, called bulk endocytosis, involves reinternalizing large regions of the cell plasma membrane to generate large compartments called vacuoles, from which new synaptic vesicles eventually form. This mechanism has been observed when neurons fire at high frequency. The cellular processes underlying bulk endocytosis are not well understood, although several studies suggest proteins called dynamins are important.Wu et al. simulated the conditions a cell experiences during high levels of activity in neurons that lacked the two major dynamins present at the synapses between neurons—dynamin 1 and dynamin 3. In these neurons, robust bulk endocytosis occurred, suggesting that these two major neuronal dynamins do not play a role in this process. Furthermore, formation of vesicles from the vacuoles generated by bulk endocytosis appeared to be clathrin-independent. These findings point to the occurrence of a pathway of synaptic vesicle recycling that bypasses the need for dynamin 1 and 3 as well as for clathrin.To reconcile these results with previously published work, Wu et al. propose that dynamins may only be required for processes that also require clathrin. But how are vesicles recycled during bulk endocytosis if dynamins are not involved? There are currently few leads to base alternative mechanisms on. Further work is required to unravel this mystery, and to provide insights into how clathrin-dependent and independent recycling processes are linked during high neuronal activity.DOI:http://dx.doi.org/10.7554/eLife.01621.002 Synaptic vesicles (SVs) are the specialized organelles that store and secrete non-peptide neurotransmitters at synapses. Following exocytosis, their membranes are rapidly recaptured by endocytosis and recycled for the generation of new fusion-competent SVs . Strong One proposed alternative recycling mechanism is so-called ‘kiss-and-run’, a process whereby SVs fuse only very transiently with the plasma membrane without collapsing into it, so that the SVs reform directly by the immediate closure of the fusion pore . Yet anoCaenorhabditis elegans have opened new questions on SV recycling mechanisms. Mutations expected to result in loss or strong defect of the function of clathrin or of its endocytic adaptor AP-2 were shown not to abolish synaptic function   . Subsequriginate  challengndosomes .The goal of this study was to gain new information into the recycling of SV membranes via bulk endocytosis. To this aim we examined the impact of manipulations that affect dynamin-dependent endocytosis on this process using electron microscopy (EM) in combination with endocytic tracers. We demonstrate that the rapid, bulk endocytic recapture of SV membranes induced by a strong stimulus, either KCl or high frequency stimulation, is not impaired at synapses lacking both of the two predominant neuronal dynamins, dynamin 1 and dynamin 3, that is synapses where CME is severely perturbed . Convers+-induced depolarization results in a prominent burst of bulk endocytosis even in the absence of dynamin 1  , dynamin 3 KO (Dyn3 KO) and dynamin 1 and 3 double KO (Dyn1/3 DKO) mice. In order to reduce the contribution of genotypic variability, comparisons were made between cultures derived from littermates: (1) Dyn1 KO mice and WT mice within the litters obtained from the breeding of DNM1+/− mice and 2) Dyn1/3 DKO mice and Dyn3 KO mice within the litters obtained from the breeding of DNM1+/−; DNM3−/− mice.A previous study had suggested that stimulation of SV endocytosis by high extracellular KM1 gene) . To asse+ channel blocker tetrodotoxin  nearly completely reverses this phenotype, as lack of dynamin 1 and 3 only severely delays, but does not completely block CME (2+ to prevent SV exocytosis) prior to stimulation. Cultures were then stimulated for 90 s with 90 mM KCl to evoke maximal SV exocytosis and rapidly washed and further incubated in resting buffer (see below) for the analysis of SV recovery. To visualize endocytic organelles, cholera toxin-horseradish peroxidase (CTX-HRP) was added to the cultures both during stimulation and during the recovery. This probe binds the ganglioside GM1 on the cell surface and is an efficient marker of endocytic membranes (Spontaneous network activity drives the accumulation of endocytic clathrin coated pits (CCPs) in cultures of Dyn1 KO or Dyn1/3 DKO neurons . Howeverlock CME . Thus, iembranes .2 of synapse cross-section) was reduced to about 20% of the starting value  represents the global SV pool that is present in nerve terminals before the stimulus, and which undergoes massive exocytosis with the stimulation. The sector delineated by a dotted line reflects the pool of SVs that was still missing at the time point of recovery examined, revealing a delay in SV reformation in the absence of the neuronal dynamins. The white sector of the pie represents SVs that are unlabeled by CTX-HRP. In spite of the absence of HRP reaction product, even these SVs are likely to be of endocytic origin given the complete depletion of SVs produced by the stimulus. As stated above, these vesicles are most likely unlabeled due to the less than 100% efficiency of labeling, as supported by the observation that the size of this pool roughly correlates with the number of recovered SVs. The black sector shows the CTX-HRP-labeled SVs reformed during the 90 s stimulus, while the striped sectors represent the labeled SVs generated during recovery . Within the striped sectors, the portion with red stripes represents the fractions of SVs that become labeled when CTX-HRP is added only after the stimulus , thus implying that the portion with black stripes represents SVs that form from bulk endosomes generated during the stimulus. Our conclusions from these comparisons are as follows: (1) SV reformation occurs even in the absence of Dyn1 and Dyn3, but this process is substantially delayed as shown by the missing portion of the pie; (2) No SV reformation occurs during the stimulus in the absence of Dyn1 or both Dyn1 and 3 (solid black sections), leading to complete SV depletion; (3) Reformation of SVs by endocytosis that occurs after the interruption of the stimulus is surprisingly similar in all genotypes (red stripes sector); (4) Most of the delay in recovery in dynamin KO neurons is due to a delay in the conversion from bulk endosomes to SVs (black stripes).From the analysis of the data presented so far, one can draw the conclusions summarized in the pie charts of To further assess the delay in the generation of new SVs from bulk endosomes and the consequences of the absence of dynamin 1 and 3 on this process, further experiments were performed that employed fluid phase HRP (rather than CTX-HRP) to follow SV recycling. While this probe labels endocytic vesicles less efficiently than CTX-HRP, as it is not bound to, and therefore concentrated at, membranes, it can be rapidly washed away from the cell surface, and thus can be used to better analyze endocytosis in a specific time window. Neuronal cultures were prepared and stimulated as described above, but with the presence of free HRP during the stimulation and with the immediate wash of HRP at the end of the stimulation.As observed in experiments with CTX-HRP, SV number was strongly decreased, but not abolished in WT and Dyn3 KO nerve terminals at the end of the stimulus , when nu+ bars in + bars), and thus derived from bulk endosomes. As in the case of experiments with CTX-HRP, one can conclude that SV reformation is blocked during stimulation and delayed during recovery, with at least a portion of this delay being explained by a delay in the conversion of bulk endosomes to SVs.Results with fluid phase HRP are summarized in the pie charts of bona fide SVs, that is that they are competent for a new cycle of stimulated secretion. To address this issue, Dyn1/3 DKO cultures, and Dyn3 KO cultures as controls, were exposed to high K+ stimulation in the presence of soluble HRP, then washed and further incubated for 30 min in resting medium in the absence of HRP, exposed to a second high K+ stimulus, and finally rested again for 30 min, always in the absence of HRP. As shown by EM analysis at the end of each period  cultures  and recovery (3 and 10 min) conditions.To assess in more detail the potential occurrence of budding intermediates on bulk endosomes under conditions that optimally preserve their native ultrastructure, we performed high pressure freezing/freeze-substitution on neuronal cultures from Dyn1/3 DKO mice and from their control (cultures from Dyn3 KO siblings) cultured on sapphire disks and exposed to resting, stimulatory  KD experiments using a lentivirus based strategy . CHC was+ buffer and finally recovery in HRP-free resting medium) with analysis by EM. Prior to stimulation, only occasional CCPs were observed at synapses of both genotypes . As expd of CHC . Howevernditions . Stimula neurons . In summIt was proposed that formation of SVs from nerve terminal endosomes could occur via AP-1 and/or AP-3-mediated pathways sensitive to brefeldin A . However+ depolarization or high frequency electrical activity) occurs predominantly via the budding of SVs from endosomal intermediates generated by bulk endocytosis (bulk endosomes). They show that formation of bulk endosomes robustly occurs in the absence of the two dynamin isoforms (dynamin 1 and 3) that collectively account for the overwhelming majority of neuronal dynamin  mutant flies at the restrictive temperature  was challenged by genetic studies in nt worms . Further elegans  and mous elegans  expressi elegans . The ende et al. , 2013b iThe mechanisms through which membranes of bulk endosomes are converted to SVs remain elusive. SVs are a highly specialized form of small vesicular carriers present in all cells. The prevailing general mechanism for vesicle budding in the secretory and endocytic pathways is the assembly on the donor membrane compartments of a ‘coat’, whose core and accessory subunits are responsible for two major functions: induction of bilayer deformation and protein cargo selection . Among tSeveral membrane-associated scaffolds that mediate budding at the surface of endosomes have been described in recent years , but nonAn important open question is why the conversion of bulk endosomes into SVs is delayed in mammalian synapses that lack dynamin 1 and 3 (our study) and in dynamin mutant worms . A direcWhile the revision of this manuscript was being finalized, a paper by DNM1−/−, DNM3−/−, and DNM1−/−; 3−/− mice was described previously  to wash out TTX, cultures were pre-incubated for 5 min with HRP  or CTX-HRP  in the same buffer. Subsequently, they were stimulated by incubation for 90 s, this time with 2 mM Ca2+, but with a high concentration of K+ (90 mM) and a corresponding reduction in Na+. Alternatively, after a rinse with Tyrode buffer, coverslips were placed into a Chamlide stimulation chamber  and neuronal cultures were subjected to 10 s electrical field stimulation at 80 Hz  using a Master-8 pulse stimulator . Cultures were then washed rapidly three times in Ca2+-free Tyrode buffer and subsequently allowed to recover under conditions that prevent synaptic activity, either Ca2+ free Tyrode buffer also containing 0.5 mM EGTA or Tyrode buffer containing 1 μM TTX. HRP or CTX-HRP were present during the stimulus, the recovery period of both, as described in the text. All procedures involving mice were approved by the Yale University Institutional Animal Care and Use Committee.Primary cultures of cortical neurons were prepared from E18 day mouse brains as described  and anal4, 1.5% K4Fe(CN)6, , in the same buffer, en bloc stained with 0.5% uranyl magnesium acetate, dehydrated, and embedded in Embed 812 . The HRP reaction was developed with diaminobenzidene and H2O2 after the glutaraldehyde fixation step. Ultrathin sections were observed in a Philips CM10 microscope at 80 kV and images were taken with a Morada 1 k × 1 k CCD camera (Olympus). Synapses were selected based on the presence of an active zone. SV number and the membrane area of HRP-labeled bulk endosomes within the 60 nm thick section (endosome perimeter multiplied by 60) were measured from 30 to 100 synapses for each condition (1–3 pups for each condition depending on the genotypes found in the litter) and experiments. These values were normalized to the cross-sectional area of the presynaptic terminal. Results of the morphometric analysis are presented as average ± SEM, with the exceptions of data shown in Cells were chemically fixed with 1.2% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% OsOElectron tomography was performed as described  on 200–22O at −90°C for 1-3 hr. Subsequently, they were slowly shifted (with temperature increases of 10°C/hr) to −20°C, kept for 12 hr at this temperature, and then further shifted to +20°C (again with temperature increases of 10°C/hr). Samples were then washed in acetone containing 1% H2O (10 min × 3), incubated with 0.1% uranyl acetate +1% H2O in acetone for 0.5–1 hr at room temperature (RT), washed in pure acetone at RT (10 min × 3) and infiltrated at RT with epon using the following sequence: 1 hr epon/acetone 1:2 (vol/vol); 1 hr epon/acetone 2:1 (vol/vol); 2 hr pure epon at RT. Finally, embedded samples were polymerized at 60°C for 48 hr.Neurons were cultured on the opposite side of 20 nm carbon-coated 3 mm sapphire discs  (scratched with a unsymmetric symbol to distinguish sidedness). For freezing, discs were removed from the culture dishes, sandwiched between the 100 μm side of an A hat and the flat side of a B hat  and flash frozen under high pressure with a Leica EM HPM100 machine with or without 20% BSA . All these manipulations were performed within 20–30 s. Frozen neurons were transferred to liquid nitrogen for storage. For freeze substitution , samplesFor CHC KD, a target sequence for mouse CHC (5′-GAAGATAAGCTGGAATGTTCT) was designed using the Block-iT RNAi Designer  and subcloned into pcDNA6.2/GW-EmGFP-miR  following manufacturer's instructions. The EmGFP-miR cassette was amplified by PCR and subcloned into the pRRLsinPPT vector to generate the lentiviral expression vector. The control virus has been described previously . VSV-G p review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  in synaptic vesicle recycling and its relationship to the clathrin- and dynamin-dependent pathway. The authors use cultured neurons that are subjected to a massive, vesicle-depleting stimulus and then allowed to recover exocytosed membranes by endocytosis, partially in the presence of extracellular tracers. Analysis is carried out by quantitative EM, with conventional fixation/staining protocols being complemented by cryo-EM following high-pressure freezing.All referees agreed that the manuscript makes an interesting contribution to a longstanding and controversial debate. However, several of us had substantial concerns, and while in the end we decided to allow for revision, the following points should be addressed:1) Stimulation by KCl is highly non-physiological and thus the intermediate structures described here may only become apparent at extreme stimulation conditions. We therefore suggest repeating at least some of the key experiments using stimulation by action potentials (at least for 2) There was considerable debate among the referees whether the remaining proteins  may not be sufficient to maintain some function despite very low protein levels. There are precedents for this: e.g. in MEFs, Parkin is only detectable by Q-RT-PCR and it is undetectable by western, yet this very low amount of Parkin is enough to support mitophagy (in parkin null MEFs mitophagy is blocked). While we realize that this issue is not easy to deal with, there should be at least an open discussion of this possibility. Also, the authors may try to carry out knockdown of dynamin 2 for further reduction of dynamin levels.3) While the authors state that synaptic vesicles are being re-formed from the endosomal intermediates, the morphological distinction between endosomes and synaptic vesicles is not clear . Also, evidence should be provided that these regenerated synaptic vesicles are capable of undergoing exocytosis.In the following, we include some of the original referee comments to explain the concerns listed above in more detail:Reviewer #1:1) (...) The conclusion that synaptic vesicles are able to recycle without clathrin or without dynamin largely stems from the observation that bulk endosomes resolve into small synaptic vesicles in dynamin 1/3 double knock outs or upon additional clathrin knock down. However, not all dynamin function is absent and dynamin 2 is still present in these experiments. The authors argue that dynamin2 is present at low levels, but is very difficult to infer function simply from protein levels. Maybe a couple of percent of dynamin 2 are enough to support the very slow vesicle recycling the authors are observing. This point is further substantiated by the fact that triple knock out neurons do not survive while dynamin 1/3 double knock outs do, indicating an important function for the dynamin 2 protein despite the fact that it is present at only very low levels. A similar argument holds true for clathrin knock down: some clathrin is still persisting; there are even a few clathrin coated pits still observed after knock down, underscoring the presence of some clathrin protein upon knock down . I under2) I don't follow the argumentation that in dynamin1/3 KOs many of the synaptic like vesicles that form during the recovery period are not HRP labeled? The authors argue that stochastic labeling of all-or-non DAB conversion could explain this observation. In controls the same issues would be at play, yet there, almost all synaptic vesicles are labeled?3) The HPF EM data shows bulk endosomes that sometimes display tubular extensions. Based on these morphological features the authors argue that these extensions my yield synaptic vesicles. The authors observe a dense coat at the tips of these extensions and claim this is not clathrin. This qualitative statement is not convincing and additional evidence that this is or is not clathrin is needed to make a convincing argument. In addition, it seems that the diameter of these structures does not match the diameter of an average synaptic vesicle.Reviewer #2:The authors suggest that synaptic vesicles form after stimulation of DKO synapses. In their analysis figures  Our data proves beyond any reasonable doubt (we use double KO mice) that dynamin 1 and 3 are not needed for bulk endocytosis; this process is actually enhanced in the complete absence (germline KO) of these two neuronal dynamins, possibly because clathrin-mediated endocytosis is impaired. In contrast, Kononenk et al based their conclusions about the requirement for dynamin 1 and 3 in bulk endocytosis on RNA interference experiments that lacked even minimal controls to establish that the observed phenotypes were due to on target effects of the shRNA. Furthermore, their conclusions are indirect, based on pHluorin experiments, and not supported by ultrastructural studies.2) While the manuscript by Kononenk et al concludes that clathrin is required for the conversion of bulk endosomes to SVs, it does not provide direct evidence for the presence of clathrin on bulk endosomes. They show that after clathrin knockdown, stimulation is followed by the accumulation of endosome-like vacuoles (bulk endosomes). In contrast to their conclusion that clathrin must be required for the conversion of bulk endosomes to SVs, this observation is equally consistent with the possibility that bulk endosomes form more extensively when clathrin mediated budding of SVs from the plasma membrane is impaired.As this other paper just appeared (June 4), we did not include it in our review of “prior knowledge”. We have made a reference to it in an Addendum at the end of our manuscript:Addendum: While the revision of this manuscript was being finalized, a paper by Response to the comments of the reviewers:We found the review to be very constructive and we have now addressed all the comments with new experimentation. The delay in responding relative to the time when we received the decision letter is explained by the long-term nature of the work involved in this research. Experiments rely on i) the breeding of mice that must be kept at the heterozygous state for the dynamin 1 allele, ii) 2-3 weeks for each preparation of neuronal primary cultures to mature and iii) a time consuming analysis by EM (morphometry and reconstruction of EM tomograms).bona fide functional synaptic vesicles; 2) quantitative analysis of endocytic intermediates following action potential stimulation in addition to high K+ stimulation; 3) new high pressure freezing experiments, providing improved morphological preservation ; 4) analysis of synaptic vesicle size. In addition 5) statistical data have been added to all the figures reporting morphometric data. New data include: 1) evidence the SVs reformed by bulk endocytosis are available for a new round of release, thus indicating that they are Two new authors have been added, Mirko Messa and Xinran Liu, as they contributed to the new electrical stimulation and tomographic experiments.Points that needed to be addressed based on the editorial letter.1) Stimulation by KCl is highly non-physiological and thus the intermediate structures described here may only become apparent at extreme stimulation conditions. We therefore suggest repeating at least some of the key experiments using stimulation by action potentials (at least for).+ stimulation. These data are now shown in revised Figure 5We performed these experiments. We stimulated dyn3KO (CTRL) and dyn1/3DKO cultures at 80 Hz for 10 s in the presence of CTX-HRP. EM analysis revealed that the bulk endocytosis and recovery occurring under these conditions had similar properties to that observed with high K2) There was considerable debate among the referees whether the remaining proteins  may not be sufficient to maintain some function despite very low protein levels. There are precedents for this: e.g. in MEFs, Parkin is only detectable by Q-RT-PCR and it is undetectable by western, yet this very low amount of Parkin is enough to support mitophagy (in parkin null MEFs mitophagy is blocked). While we realize that this issue is not easy to deal with, there should be at least an open discussion of this possibility. Also, the authors may try to carry out knockdown of dynamin 2 for further reduction of dynamin levels.We invested great effort (even before submitting this manuscript) toward the generation or “null” dynamin synapses. We have in our lab the conditional KO mice of the appropriate genotype needed to achieve this goal and we used such mice to generate conditional triple KO fibroblasts . However, we found that while mouse fibroblasts survive for weeks, even after all dynamin alleles have been disrupted and all dynamin was lost, neurons die very rapidly when all dynamin alleles are disrupted. For example, expression of Cre  in cultured dynamin 1/ dynamin 3 double KO neurons harboring floxed dynamin 2 alleles resulted in neuronal death within few days, before all dynamin 2 could disappear from nerve terminals . In principle, we could have used pharmacological inhibition to block residual dynamin, as several “anti-dynamin” drugs, primarily dynasore or dyngo, have been reported in the literature.. To comply with these comments, we have now better addressed this issue in the Discussion.However, our studies of triple KO fibroblasts demonstrated major off-target effects of these drugs, thus making the use of these drugs unreliable. Thus, we could not answer the question raised here. Similar considerations apply to clathrin as we observed neuronal death before clathrin could completely disappear. Thus, we agree with reviewer 1 that we cannot exclude that slow 'classical' clathrin-mediated endocytosis from the plasma membrane, mediated by dynamin 2, could play a role in mediating the recycling of the synaptic vesicles during recovery from stimulation in the absence of dynamin 1 and 3We have also changed the title to “A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis”.3) While the authors state that synaptic vesicles are being re-formed from the endosomal intermediates, the morphological distinction between endosomes and synaptic vesicles is not clear . Also, evidence should be provided that these regenerated synaptic vesicles are capable of undergoing exocytosis.This point deals with the more general issue of what should be defined as an endosome in nerve terminals. The word “endosome” was first introduced by Helenius et al in 1983  to define “a heterogeneous population of endocytic vacuoles through which molecules internalized during pinocytosis pass en route to lysosomes”, and in which molecular sorting occurs. The morphological, biochemical and functional heterogeneity of endosomes has become even more apparent during the last 30 years. In our study we have frequently used the terms “endosomal intermediates” or “bulk endosomes” to emphasize the unique nature of the nerve terminal compartments that form and then disappear in response to massive stimulation. Here we have used size to distinguish such intermediates from synaptic vesicles. We recognize that needs to be precisely defined in the text. We have now defined endosomes as organelles bigger than 80 nm. We have also carried out a new morphometric analysis of vesicular profiles with diameters (in the plane of the section) comprised between 20 and 200 nm at WT and mutant synapses at rest and during recovery. This analysis indicated a slightly larger and more heterogenous diameter of synaptic vesicles in dyn1 KO and dyn1/3 DKO synapses.+ stimulation. Newly formed (as defined by HRP labeling) SVs are releasable from both CTRL and dyn1/3DKO neurons.We also now provide evidence that SVs regenerated from bulk endosomes are functional by showing that SVs labeled by HRP during a 90 sec high K+ stimulation can undergo exocytosis after a 30 min recovery period upon a second round of high KAlthough we understand that the points outlined in the editorial letter (above) were the critical ones, we have also addressed to our best additional specific points raised by the reviewersReviewer #1:1) (...) The conclusion that synaptic vesicles are able to recycle without clathrin or without dynamin largely stems from the observation that bulk endosomes resolve into small synaptic vesicles in dynamin 1/3 double knock outs or upon additional clathrin knock down. However, not all dynamin function is absent and dynamin 2 is still present in these experiments. The authors argue that dynamin2 is present at low levels, but is very difficult to infer function simply from protein levels. Maybe a couple of percent of dynamin 2 are enough to support the very slow vesicle recycling the authors are observing. This point is further substantiated by the fact that triple knock out neurons do not survive while dynamin 1/3 double knock outs do, indicating an important function for the dynamin 2 protein despite the fact that it is present at only very low levels. A similar argument holds true for clathrin knock down: some clathrin is still persisting; there are even a few clathrin coated pits still observed after knock down, underscoring the presence of some clathrin protein upon knock down . I understand that it is at present not possible to generate neurons that lack dynamin (or clathrin) completely, but because of this, the interpretation that synaptic vesicles can be formed in the absence of either protein lacks experimental support and the alternative interpretation that slow 'classical' endocytosis mediates the recycling of the synaptic vesicles seems possible and even more realistic.showing suggesting that this process is independent of clathrin-mediated budding. We have modified the title and we have been very cautious throughout the text. For example the last sentence of the Introduction  states: “These observations provide evidence for a pathway of SV reformation that does not require dynamin/clathrin mediated endocytosis and that predominates after strong stimulation”.We have addressed this question within point 2 of the compiled editorial comments (see above). We agree with this reviewer that residual dynamin, dynamin 2, could play a role in the recycling occurring in the absence of dynamin 1 and 3. In fact, we do believe that it may play some role in clathrin-mediated endocytosis from the plasma membrane still occurring in these cells. However, our data argue against a clathrin/dynamin-dependent classical mechanism from bulk endosomes in dyn1/3DKO neurons, as we do not see clathrin coated pit accumulation on bulk endosomes at synapses of these neurons, in contrast to the robust accumulation of CCPs at the plasma membrane. Tomographic analysis demonstrated that CCPs localized deep into axon endings were in fact localized on deep plasma membrane invaginations. In any case, we have now changed the following sentence of the Abstract by replacing “showing” with “suggesting”: “Conversion of bulk endosomes into SVs during recovery was delayed, but not blocked, even after further clathrin knockdown, 2) I don't follow the argumentation that in dynamin1/3 KOs many of the synaptic like vesicles that form during the recovery period are not HRP labeled? The authors argue that stochastic labeling of all-or-non DAB conversion could explain this observation. In controls the same issues would be at play, yet there, almost all synaptic vesicles are labeled?There is some heterogeneity in the fraction of labeled SVs, but, as the quantification data show , such fr3) The HPF EM data shows bulk endosomes that sometimes display tubular extensions. Based on these morphological features the authors argue that these extensions my yield synaptic vesicles. The authors observe a dense coat at the tips of these extensions and claim this is not clathrin. This qualitative statement is not convincing and additional evidence that this is or is not clathrin is needed to make a convincing argument. In addition, it seems that the diameter of these structures does not match the diameter of an average synaptic vesicle.We thank the reviewer for this comment. Those tubular structures were indeed very rare and could not be observed in new samples that we have generated with improved morphology. All high pressure-freezing/freeze-substitution images presented are new. The only coated buds observed in vacuolar structures had the characteristic texture of clathrin-coated pits, but these were very few , the authors present separate graphs for bulk endosomes and synaptic vesicles. It is not clear how the authors decided whether a specific organelle is a synaptic vesicle or a bulk endosome. (…) The authors should therefore include a characterization of the sizes of the vesicles forming during recovery after stimulation, and should indicate the amount of such vesicles that are within the size range of normal, WT synaptic vesicles.Based on these measurements, a clear cutoff should be defined between synaptic vesicles and bulk endosomes .This question was answered above.Along the same lines, the authors need to determine whether the small vesicles forming in DKO synapses exhibit the physiological characteristics of synaptic vesicles. For example, can these vesicles exocytose upon stimulation? If not, then this pathway cannot be suggested to form true synaptic vesicles. A number of strategies could be tried here, including pHluorin experiments and FM dye uptake and release.We addressed this question with a new experimentation (as answered above), and found that the vesicles can undergo exocytosis.


During the outbreak, 50% of household members living with an infected person developed symptom of monkeypox infection. >1 transmission event, and >6 transmission events occurred within this health zone. Mean incubation period was 8 days (range 4–14 days). The high attack rate and transmission observed in this study reinforce the importance of surveillance and rapid identification of monkeypox cases. Community education and training are needed to prevent transmission of MPXV infection during outbreaks.A 600-fold increase in monkeypox cases occurred in the Bokungu Health Zone of the Democratic Republic of the Congo during the second half of 2013; this increase prompted an outbreak investigation. A total of 104 possible cases were reported from this health zone; among 60 suspected cases that were tested, 50 (48.1%) cases were confirmed by laboratory testing, and 10 (9.6%) tested negative for monkeypox virus (MPXV) infection. The household attack rate  was 50%. Nine families showed  Orthopoxvirus, is zoonotic and endemic to western and central Africa. MPXV is a close relative of the variola virus, and monkeypox illness resembles a smallpox-like infection but is less severe than smallpox. Most patients initially develop a fever, followed by rash a few days later. Lymphadenopathy is a common sign and occurs just before or concomitant with the rash , which belongs to the genus ,,,,MPXV transmission among close contacts within households is well documented; previous reports have shown up to 6 intrafamily transmission events (Monkeypox is a reportable disease in the Democratic Republic of the Congo (DRC), and cases are reported from 26 health districts . During 2013, a substantial increase in the number of suspected human monkeypox cases was noted in the Bokungu Health Zone within Tshuapa District of DRC’s Equateur province . In DeceDRC has a regional surveillance system that collects reports of all suspected monkeypox cases. When possible, cases are investigated, a monkeypox-specific case report form is completed, and replicate diagnostic specimens (derived from lesions) are collected. During this investigation, additional retrospective cases within affected villages and households were identified on the basis of physical symptoms that were reported by patients and family members but that had not previously reported to the surveillance system.>1 of the following 3 characteristics: 1) rash on the palms and soles, 2) lymphadenopathy, 3) fever preceding rash. In addition, this person has a PCR-tested diagnostic specimen that yielded a positive result for Orthopoxvirus or had MPXV DNA signatures. A probable case occurred in a person with a history of high fever, a vesicular-pustular rash, and >1 of the following 3 characteristics: 1) rash on the palms and soles, 2) lymphadenopathy, 3) fever preceding rash. In addition, this person has a history of contact with a person or animal with confirmed monkeypox during the 14 days before illness onset. A suspected case occurred in a person with a history of high fever, a vesicular-pustular rash, and >1 of the following three characteristics: 1) rash on the palms and soles, 2) lymphadenopathy, 3) fever preceding rash.Cases in our investigation must have occurred during July 1–December 8, 2013. We used the following case definitions as part of the enhanced surveillance system in Tshuapa District. A confirmed case occurred in a person with a history of high fever, a vesicular-pustular rash, and 2 test by using STATA 14.0 (https://www.stata.com/). Age was considered dichotomous because vaccination was available only to those >33 years of age, as a result of the discontinuation of smallpox vaccination in DRC (and other countries) 33 years earlier (1980) after the announcement of smallpox eradication.Clinical symptoms were documented by a trained healthcare provider or investigator who used a standardized case-reporting form. Smallpox vaccination status was documented on the basis of patient recall and presence of a vaccination scar on the left upper arm. Analysis of vaccination status was performed with χOrthopoxvirus-specific real-time PCR assay –specific DNA .Diagnostic specimens  were collected and shipped to the Institut National de Recherche Biomedicale in Kinshasa, DRC, for analysis. DNA was extracted from each specimen, and an ,,Incubation period was defined as the number of days between contact with a symptomatic monkeypox patient and development of rash. Rash was chosen as the benchmark of infection for estimating incubation periods because families were better able to recall the day of rash onset than to recall the day of fever onset. To obtain the best estimate of the MPXV incubation period, we identified patients who reported clear dates of exposure and rash onset in our investigation and in the published literature  within families and villages on the basis of the calculated incubation periods for household transmission. Cases were considered independent when the interval between the onset of rash for a case-patient in the household or village was >8 days from the onset of symptoms for the previous case-patient. This value was chosen because we assumed that the first case occurred after the shortest possible incubation period (5 days) and that the last possible case occurred after the longest possible incubation period (13 days). Any cases occurring after this window of 5–13 days are considered to result from an independent infection either inside or outside the household. Coordinates for case households were recorded with handheld global positioning system units  and compiled with the locations of residential structures digitized from satellite images . Household counts were aggregated into a 50-square-meter grid covering the entire populated area. Tests for spatial autocorrelation were performed by using the Global Moran’s I tool in ArcGIS 10.2  at distances of 50–1,000 meters in 50-meter increments.During 2013, a total of 104 suspected case-patients with human MPXV infection and 10 deaths (9.6%) were reported from the Bokungu Health Zone to the national surveillance system, with October showing a dramatic increase in number of cases . Of the During the focused investigation period (July–December 2013), we identified and interviewed 63 case-patients in 16 households . Of thes2 analysis showed that that this difference was not significant (p>0.05). However, vaccination status and age >33 years were nearly perfectly correlated (p< 0.001).In the 16 investigated households, 9 (15%) of affected household members had evidence of a prior smallpox vaccination, compared with 30% of unaffected household members; χThe median number of persons affected within each household was 3 . The median attack rate within households was 50%; mean was 52.1% (range 50%–100%). For 1 of the 16 families investigated, all 6 household members were affected. For all households, the median interval between the time that rash developed in the first person in the household to time that rash developed in the last person was 10 days (range 2–41 days).http://www.ncbi.nlm.nih.gov/pubmed) search identified 12 additional persons who had confirmed or probable infection and well-defined incubations periods; these case-patients had an incubation period of 9–14 days , an additional 4 households showed patterns consistent with transmission within the household  was used, 9 of 16 households showed ousehold , panel Aousehold , panel Bnt cases , panel CHuman MPXV infection is endemic to DRC, with cases occurring throughout the Congo Basin. Many of these cases occur in isolation or in small clusters, but large outbreaks occasionally occur that involve many persons over a large geographic area. During 2013, a total of 104 cases of human monkeypox illness were reported in the Bokungu Health Zone. In contrast, only 17 cases were reported in 2011 and 13 in 2012. The surveillance system did not change substantively during this period; consequently, the rate in 2013 represents an increase of >600%, compared with rates for previous years. Our investigation focused on cases that occurred during the height of the 2013 outbreak.http://esa.un.org/unpd/wpp/), 17.8% of the population in DRC is <5 years of age. Consequently, the age demographics of patients during the monkeypox outbreak correspond to those of the general population, suggesting that young children are not more prone to MPXV infection than others. A parallel study performed during this outbreak found no association between monkeypox illness and hunting or consumption of specific animals , and this period was dependent on the route and nature of exposure . These clusters could have been created in 3 different ways. First, the clusters could be linked sequentially, whereby the infection could be externally introduced into a cluster and then passed by 1 person from that group to cluster 2 and so on. Second, transmission may not have occurred in a clear linear fashion, but persons may have had multiple human exposures. Third, MPXV could have been reintroduced into the community from an external source (zoonotic or human) during the course of the outbreak. Although we cannot determine which of these possibilities is most likely, we favor the second model because community interactions would make a strictly linear pattern of spread unlikely to occur. Further, the limited number of cases in the population as a whole makes it less likely that an external source was causing frequent reintroductions. We can conclude that >6 transmissions or introductions occurred in this health zone after the initial infection.Altogether, the Bokungu Health Zone had 42 cases in ,This report has limitations that should be considered. First, MPXV infection was laboratory confirmed in 48% of the cases by using PCR; the remaining cases were identified by patients’ symptoms. Laboratory confirmation was not possible for many cases because patients were interviewed after symptoms had resolved. Local resources for performing specimen collection were unavailable during all phases of the outbreak, so confirmation of MPXV infection for many cases was not possible. The lack of specimen collection has been noted as a limitation of the current surveillance program, and we are actively addressing this issue. Second, modeling of the incubation period was limited by the inability of most patients to identify a specific source of infection or a date of exposure. We assumed a minimum incubation period of 3 days when we created the incubation period model. This assumption was necessary to prevent bias toward very short incubation periods and is appropriate because of the longer incubation periods observed in animal models (This analysis provides insight into the dynamics of MPXV infection. We observed an average household attack rate of 50%, a much higher rate than reported in previous studies. Measures to decrease this attack rate should be implemented, including family-based education related to hygiene and isolation of patients. The transmission patterns observed in this outbreak also suggest transmission at the community level; therefore, community-wide education should begin as soon as the first monkeypox case is identified in an area. The calculated incubation period of 5–13 days further refines our understanding of the longest period of MPXV transmission risk after exposure in a natural setting. Knowledge of transmission risk is helpful for considering the appropriate monitoring period for exposed persons. This investigation and future work will improve our understanding of MPXV infection and our ability to limit its spread.


The lipothioureas were shown to reduce non specific interactions with cell membranes and deliver their DNA content intracellularly more efficiently, as compared to cationic lipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targeting in vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and less accumulation in the lungs after an intravenous injection in mice. They also induced luciferase gene expression in muscle and tumor after local administration in mice. Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes as transfecting agents. In this review we will focus on the structure activity studies that permitted the identification of the two most efficient thiourea lipids.Non-viral gene therapy requires innovative strategies to achieve higher transfection efficacy. A few years ago, our group proposed bioinspired lipids whose interaction with DNA was not based on ionic interactions, but on hydrogen bonds. We thus developed lipids bearing a thiourea head which allowed an interaction with DNA phosphates through hydrogen bonds. After a proof of concept with a lipid bearing three thiourea functions, a molecular and cellular screening was performed by varying all parts of the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Two lipothiourea-based structures were identified as highly efficient  Non-viral vectors have been investigated particularly against the cystic fibrosis disease which has allowed progress on both transporters and plasmids [Non-viral gene therapy is an attractive strategy to provide gene transfection while avoiding the safety concerns raised by viral vectors. Non-viral vectors are synthetic compoundplasmids ,2. More plasmids . Concernplasmids .Most gene delivery systems use electrostatic interactions to complex DNA and exhibit a cationic character. Unfortunately, this positive charge enhances non-specific interactions and therefore, cationic vectors cannot be strictly envisioned to specifically target tissues. This problem has usually been dealt with by masking the cationic surface charge , but rarIn our search for an alternative system, we envisioned that a new approach based on hydrogen bonding could provide gene protection and reduce unspecific interactions. Indeed, the tertiary structure of biomacromolecules is based on different types of non-covalent interactions, including hydrogen bonds. Although hydrogen bonds are weak, they are able to maintain a complex tridimensional structure, as evidenced by the double-stranded helix structure of DNA. We hypothesized that the multi-hydrogen bonding provided by a supramolecular assembly would induce a cooperative effect leading to DNA condensation. For this purpose, we chose the thiourea function, known to be the strongest neutral hydrogen bond donor to carboxylates and phosphates.The thiourea function is similar to a urea function, in which the oxygen atom has been replaced by a sulphur atom. The two amino groups surrounding the C=S linkage strengthen the electronegativity of the sulphur atom, enhancing its capacity to form hydrogen bonds . ThioureConsidering the above mentioned advantageous properties of the thiourea moiety, we therefore chose to incorporate this function at the polar head of a lipid, in order to form lipothiourea-based liposomes . We coulFor the purpose of this review, we will focus on neutral lipothioureas, for which interactions with negatively charged serum proteins and proteoglycans are expected to be greatly reduced. This review summarises the evolution of these lipids from the first prototype to the current well-characterized and biologically active lipothiourea lipids.2.2.1.2.1.1.The first thiourea-based lipid synthesized, called DT3TU, bore three thiourea functions, as shown in Despite this major drawback, DT3TU could be formulated with dipalmytoylphosphatidylcholine (DPPC) or PEG-lipid to avoid aggregation, and obtain a formal proof of concept of the maintained thiourea function capacity to interact with DNA in its lipidic form . When as2.1.2.A systematic structure-activity relationship was performed to establish the crucial structural points of the new lipids. The four lipid components, namely the hydrophobic anchor, spacer, linker and polar head extremities were thoroughly evaluated . The geoWe first evaluated the solubility of the compounds in an aqueous medium, in ethanol or in chloroform, to choose the appropriate method to form a suspension with these lipids in an aqueous medium, either hydration of a lipidic film or ethanolic injection. LogP was calculated for all the lipids, as summarised in 2.1.3.5 n = 4; not represented). The isopropylidene protecting groups were then hydrolysed with aqueous HCl to give the final products 1, 2 and 3.The selected compounds have been obtained on a larger scale thanks to an optimised chemical process. The general synthesis is described in Scheme 1. Briefly, the dialkylamines were reacted with diglycolic anhydride in dichloromethane to give the corresponding carboxylic lipids. 1,3-diazidopropan-2-amine was obtained in three steps from serinol . Condens6–8, the dialkylamines were condensed with Boc-N-protected lysine using BOP as the activating agent. The protecting groups were subsequently cleaved by the action of trifluoroacetic acid, and the resulting diamino derivatives were reacted with the isothiocyanate to give the protected thioureas 6, 7 and 8. It should be pointed out that deprotection of the diol groups was not possible in this series, due to the Edman degradation of the α-N-thiourea function [To obtain compounds function . Neverth2.1.4.The number of thiourea functions has been reduced from three to two between the first thiourea lipid synthesized and the series of lipids described above . We showC10SDigLSerPdiol compound , reduced the efficiency of the interaction with DNA for the serinol series. The opposite was found for the lysine series formulated with DPPC which interacted more efficiently with DNA by increasing the lipid length. We will see next that this has a dramatic impact on the transfection efficiency.The ability of most of the lipids to interact and associate DNA was evaluated by different methods. Gel retardation assays gave the thiourea/phosphate ratio required to fully retain DNA as indicated in 3, 4, 5 in 5 to 3), increased the lipothiourea/DNA interaction efficacy. To further study the interaction occurring between DNA and lipothiourea assemblies, we performed a thorough NMR study by synthesizing a 13C-enriched compound 3. This study indicated that the last deprotection step corresponding to the isopropylidene hydrolysis in acidic medium was crucial for this series. Deprotection led to compounds of similar molecular mass, but with either an iminothiol or a thiourea function depending on the strength of the acid used (strong hydrochloric acid or weak acetic acid). The presence in liposomes of the iminothiol which can only form and remain stable in drastic conditions could only be explained by the favoured hydrogen bonds of the lipidic auto-assembly [This study also evidenced the influence of the chemical function at the polar head extremity. Comparing compounds assembly .2.2.Using cryo-transmission electronic microscopy, we showed that lipothioureas self-assembled as unilamellar liposomes following ethanolic injection of low concentration in water 2.3.2.3.1.in vitro. Apart from compound 5, all lipids tested transfected cells with various efficiencies  were compared, and we noted a clear dose-dependent increase in transfection for both families of lipids 3, (AC10SDigLSerPdiol), and compound 6 (AC14LLysPdiolP) which were found to be the best transfecting agents from their series, are also the lipids which showed the strongest interaction with DNA [Another point of interest is the relationship between lipid/DNA interaction and transfection efficiency. The two lead candidates, compound with DNA . In addi2.3.2.3 to transfect cells with DNA, we thought that this lipid could be applied as a siRNA carrier. We firstly chose to evaluate the feasibility of siRNA delivery by lipothiourea by simply mixing thiourea lipid and siRNA , whereas in the case of thiourea lipoplexes, a clear membrane localisation is observed (b). When both lipids and DNA are labelled, one can clearly observe that DNA (green spots) is more efficiently released from lipothiourea (d) than from cationic lipoplexes (c). Collectively, these results could explain why complexes, which are poorly internalised by the cells, are transfecting cells efficiently. Thiourea lipoplexes have also been shown to be internalised mostly by a caveolae process while DMAPAP lipoplexes would be mostly internalised via clatrin endocytosis [Following the release of fluorescently labelled DNA into the cells, we also found that thiourea lipids released more efficiently their DNA content . The weaocytosis . This di2.3.4.The initial interest in avoiding cationic charges was to allow cell targeting by reducing unspecific cell interactions. Besides, grafting a targeting ligand at the surface of thiourea liposomes could increase the amount of lipoplexes internalised by specific cells.vβ3 integrin ligand by means of a lipid-PEG to target endothelial cells. Addition of a RGD-PEG2000-lipid or RAD-PEG2000-lipid at the surface of lipothiourea complexes allowed obtaining a higher specific transfection efficiency comparing RGD and RAD ligands as shown in 2000-lipid from 0.5 to 1% decreased the gene expression, probably due to a PEG shielding effect, which could reduce the internalisation of the lipoplexes into the cells [To this end, we added an αhe cells . Despite2.4.2.4.1.3in vitro, we next investigated the potential application of thiourea lipoplexes in vivo in mice. In vivo, intramuscular and intratumoral administrations of thiourea lipoplexes have been evaluated using luciferase expression. Thiourea lipids did not inhibit DNA transfection mediated by electrotransfer in the muscle, as found with charged delivery systems [Given the good transfection results obtained with compound  systems . Moreove systems .2.4.2.3 and 30% of a thiourea-cholesterol derivative. The lysine derivative 6 was tested in presence of DPPC as formulated for in vitro studies.The biodistribution of thiourea lipoplexes after intravenous administration was compared to conventional liposomes made with phospholipids. The composition of thiourea liposomes was similar to that of conventional liposomes , as theyResults presented in 6 showing the reduction of unspecific interactions. These results confirmed our hypothesis that neutral lipids could be more interesting than charged lipids for in vivo gene delivery.Comparing lipothiourea with cationic lipoplexes, we found that the amount of thiourea lipoplexes was significantly higher in the blood 2 h post-injection 3.In summary, we have designed original neutral transfecting vectors containing thiourea functions. The introduction of two thiourea functions in the lipid allowed the compaction and protection of DNA. A rigorous structural activity relationship has been performed to determine the impact of each part of the lipid. We found that the lipid length and the polar head were the parts which impacted the most both formulation and transfection.in vitro, even though lipothiourea were poorly internalised by the cells. An efficient release of the nucleic acids in the cells as well as a major caveolae internalisation pathway could explain the efficacy of this transfection.We showed that thiourea lipids were able to transfect cells either with DNA or siRNA in vivo was increased as compared to that of charged systems. The level of transfection was still poor, as obtained with cationic lipoplexes, but targeting could improve transfection in vitro and should be evaluated in vivo. Circulation time of lipothiourea was similar to the one of conventional liposomes, which are currently clinical use for doxorubicin delivery. This is rather interesting as lipothiourea present the advantageous properties over conventional liposomes to condense, protect and transfer DNA into the cells. Further in vivo evaluations on relevant models should be performed with adapted formulations including PEG or cholesterol to further increase the lipidic auto-assembly stability.Finally, the amount of thiourea lipoplexes recovered in the blood