Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3′ motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interactive viral ecosystem with sequence variability ranging over 2 orders of magnitude and evidence of recombination, horizontal gene transfer and variable fragment numbers. Large numbers of viral RNAs were detected in multiple Agaricus samples; up to 24 in samples symptomatic for disease and 8–17 in asymptomatic samples, suggesting adaptive strategies for co-existence. The viral composition of growing cultures was dynamic, with evidence of gains and losses depending on the environment and included new hypothetical viruses when compared with the current transcriptome and EST databases. As the non-cellular transmission of mycoviruses is rare, the founding infections may be ancient, preserved in wild Agaricus populations, which act as reservoirs for subsequent cell-to-cell infection when host populations are expanded massively through fungiculture.Thirty unique non-host RNAs were sequenced in the cultivated fungus,  Vitis vinifera, associated with the disorder Syrrah/Shiraz decline2 and multiple virus infections have been described in insects such as the honey bee4. While in Homo sapiens infection with single viruses is mostly associated with a single pathogenic outcome, there is evidence that multiple viral infections can change disease symptoms7. In fungi, infection has been associated with a large numbers of distinct RNAs8: 26 RNAs have been identified in the fungus Beauveria bassiana with 11 in a single isolate9, and 26 RNAs found in the cultivated mushroom Agaricus bisporus with 16 in a single isolate10.Multiple viral infections occur in many Eukaryotic organisms, animals, plants and fungi. Seven RNA viruses have been sequenced from the grapevine, initial infections from free non-cellular viruses have been difficult to demonstrate with only one example, a DNA virus, of fungal infection11. Initial infections are therefore likely to be rare and ancient events which, once established, lead to long-term co-existence between virus and host in a non-lethal persistent life-style. Furthermore fungal growth, which is characterised by cytoplasmic exchange in the vegetative and sexual phases, promotes the accumulation of multiple viruses which then develop coexistence strategies within an individual hypha, colony or hyphal network.Multiple viral infections are often asymptomatic representing persistent static life-styles which appear to be benign, symbiotic or possibly beneficial to the host. For fungi, viral infection occurs predominantly via cytoplasmic exchange following cell-to-cell contact and 12. Horizontal gene transfer has been inferred between positive-sense RNA viruses from different families in the fungal pathogen, Sclerotinia sclerotiorum13. At the viral population level, Syller14 described different synergistic and antagonistic multiple viral interactions in plants leading to various outcomes, such as pathogenesis, viral evolution and the spatial separation of viruses among plant tissues. Host factors rather than viral titre or order of infection appear to have the biggest influence on the equilibrium levels of introduced viral strains to Citrus trees15. However, the concept of ‘quasispecies’ suggests that such equilibria can also be disrupted by changing environments. The quasispecies concept describes a “cloud” of viral sequences able to adapt by population and genetic change (mutation or selection of variants) leading to changes in viral-host interactions on the spectrum from symbiosis to pathogenicity18.Multiple virus infections involve numerous interactions among the viruses themselves, the host and the environment, resulting in changes at both the molecular and population levels that can lead to a transition in life-styles from persistent to acute, resulting in a disease phenotypeAgaricus bisporus, whose genome was sequenced in 201219, offers a model system for studying the interactions of multiple viral infections and transitions in viral life-styles. A. bisporus is commercially cultivated via vegetative propagation as a high value agricultural crop, the cultivated white mushroom, with an annual production value of $4.7bn20. A. bisporus can suffer from economically-damaging viral diseases such as La France disease and Mushroom Virus X (MVX) disease. La France disease has symptoms of growth retardation and distortion of fruitbodies and is associated with identifiable particles and a 9 segment virus, AbV122. MVX disease is a collective name for a mixture of symptoms  associated with 30 dsRNAs, 26 found by Grogan et al.10, and an additional four by Eastwood et al.23. These RNAs are assumed to be unencapsidated viral genomes as no viral particles have been observed, although Romaine et al.24, found evidence of RNA virus packaging in membrane vesicles. Profoundly different levels of the low molecular weight RNA species (>103 fold difference) have been observed in adjacent non-symptomatic and diseased A. bisporus fruitbodies attached to the same mycelial network, indicative of a spatially-separated, viral life-style transition, persistent to acute23.The fungus, 25, while three viruses isolated from the Dutch Elm Disease fungus Ophiostoma novo-ulmi were found to be related26. Only two published RNA sequences have been associated with the MVX complex; the 14.5 kbp dsRNA identified as an Endornavirus27 and the partial sequence of a 17 kbp dsRNA hypothesised as a hypovirus28. Two further RNAs (1.8 and 2.0 kb) have been suggested to derive from a Partitivirus on the basis of their molecular size and apparent bipartite nature23.The extent to which the sequences of the 30 viral RNAs of MVX are related, for example as defective or satellite RNAs, remains unknown. Two reports have presented evidence of hybridisation of two or more RNAs of the MVX complex with a single probeA. bisporus will enable a greater understanding of their relatedness, interactions and dynamics, host adaptations and viral life-style transitions. In this study 30 different viral RNAs from A. bisporus mushroom fruitbodies were sequenced, characterised and classified phylogenetically. To our knowledge this is the first de novo deep sequencing study of a large number virome for a single organism.Sequencing and phylogenetic classification of the multiple viruses associated with de novo assembled. Contig structures were confirmed by PCRs spanning the full length of the contigs and Sanger sequencing of select regions as well as RACE PCR and Sanger sequencing of the 5′ ends. These analyses revealed 30 distinct RNA molecules not found in the A. bisporus genome which ranged in size from 0.5 kb–14.5 kb . Protein domains associated with RNA viruses of fungi and plants were found in 22 contigs .We propose these RNA molecules represent 18 distinct viruses based on the presence of RdRp domains in each contig (Table 1: AbV6 consisting of AbV6 RNA 1 (C2) with an RdRp domain and AbV6 RNA 2 (C12) with a capsid-like domain with homology to Tobacco Mosaic Virus (TMV); and2 AbV16 consisting of four separate contigs, AbV16 RNA 1 (C22) containing an RdRp domain, AbV16 RNA 2 (C20) containing a Vmethyltransferase, and AbV16 RNA 3 (C29) and AbV16 RNA4 (C33) each with open reading frames of unknown function. Both viruses contained shared motifs in their 3′ UTRs, no 5′ UTRs shared motifs were found in any of the sequenced RNAs.Sixteen of the contigs were consistent with monopartite viruses. In addition we hypothesise two, novel, segmented virusesWe have assigned C13, C14 and C18 to represent a single viral molecule, AbV9, however the evidence for this is incomplete and it may represent a segmented virus. C13, C14 and C18 contained different domains  , Narnaviridae, Barnaviridae, Bromoviridae, Endornaviridae, Virgaviridae, the unassigned genera, Benyvirus and Ourmiavirus and a proposed new family, Ambsetviridae (below).The RdRps of the 18 viruses have closest amino acid homology to diverse positive sense single-stranded RNA (ss(+)RNA) viral orders/families/genera: However, phylogenetic analysis based on comparisons of RdRp sequences with known viruses identified only two viruses that could be readily assigned to known viral families, and a further two formed a clade with a single unassigned hypovirus-like virus. The majority are only distantly related to known viruses, and accordingly new phylogenies are proposed , AbV10 (7.03 kb) and AbV11 (6.98 kb) (Table 28 and had 28% sequence identity at the protein level to Cryphonectria hypovirus 2 (CHPV2).Three viruses were identified with significant similarity to members of the ly, AbV2 4.6 kb, ASeveral different variants of AbV2 were detected differing in their 5′ end sequences Fig. . We defi31  to the unassigned Fusarium graminearum dsRNA Mycovirus-1 (FgVDM1). The inclusion of AbV2, AbV10 and AbV11 in phylogenetic analyses enabled an enhanced classification of the Hypoviridae . The Narnaviridae are amongst the simplest of RNA viruses consisting of a single, short replicase polyprotein (2.3–3.6 kb). There are two genera within the family, the Mitovirus and Narnavirus, with different subcellular locations, mitochondrial and cytosol respectively32.Three novel viruses were identified as putative members of the 33 .AbMV1 had closest homology to Mitovirus AEF-2013, a presumed fungal mitovirus isolated from a dipteran fly species33 Table  similar Narnaviridae has been revised by the inclusion of three new A. bisporus viruses , six hypothetical viruses identified here from the TSA databases and six more described by Cook et al.33 , AbV3, AbV5, AbV6, AbV7, AbV9 and AbSV  to the unassigned ss(+)RNA virus OMSV, Oyster mushroom spherical virus RNA viruses infecting plants and fungi with genomes of 5.4 kb–9 kb, the structure of which places them within the alpha-like super group of viruses.A. bisporus viruses in the Tymovirales have large replication ORFs of size range 5.9 kb–8.2 kb, with each containing Vmethyltransferase, protease C21, viral helicase and type II RdRp domains characteristic of members of the alpha-like supergroup. A majority also contained additional domains  with high similarity to the C14 component (RdRp) of AbV9). This places the viruses into three new clades within the Tymovirales order, tentatively named Readiviridae, Emraviridae, Teagaviridae   Table .Figure 3AbV6 and strain AbV6-2990 both contain zinc-finger RING domains (Pfam 13920) at the N terminus. AbV3 and AbSV contained a tymovirus cysteine endopeptidase as well as an N terminal DEXDc helicase while AbV5 had a domain similar to a ribosomal RNA processing protein (PF08524).Benyvirus genus within the Rubi-like virus grouping .Initial phylogenetic analysis of the RdRp domain of AbV16 RNA 1 was unable to place it consistently within any clades of the alphavirus-like supergroup. A BLASTP search of the TSA and EST databases using the translated ORFs revealed sixteen sequences with high similarity to AbV16 RNA 1 Table . Nine ofWe identified eight further non-host RNAs ranging in size from 0.5 kb to 5 kb with potential open reading frames of greater than 250 bases  and a transcript fragment identified by Eastwood Agaricus bisporus by the discovery of 18 RNA viruses and 8 ORFans by deep sequencing. The viruses are phylogenetically distinct, the majority with low similarity to any virus previously described and were neither satellite nor defective RNAs. They represent the most extensive collection of unrelated multiple viral infections yet described, the closest comparable example being for a single diseased isolate of Ophiostoma novo-ulmi which contained 12 viral RNAs, all related mitochondrial viruses32.Multiple virus infections have been identified in mushroom fruitbodies of A. bisporus cultures grew and thrived, extracting nutrients from a complex substrate and producing fruitbodies, suggesting effective co-existence strategies and adaptive mechanisms. Samples 003, 004 and 1497 displayed only mildly debilitating  symptoms of fruitbody browning while harbouring 17–24 viral RNAs (viruses and ORFans)  mechanisms19 and RNAi has been demonstrated in A. bisporus38. It is possible that some ORFs encode viral suppressors of RNAi but the identity and nature of any suppressors were not revealed by this research. However, the viral genomes contained many peptidases, numerous ORFs of unknown function and several domains of cryptic function (e.g. RING domain on AbV6 and rRNA processing on AbV5).Despite the multiple viral infections, the A. bisporus RNAs as double-stranded28 and the intended specificity of the purification procedures. It is possible therefore that these viruses have secondary and tertiary structures that enable selective binding to cellulose and protection from S1 nuclease. Three of the viruses have domains with similarities to capsid proteins , although no viral particles have been observed in MVX-infected tissues10. Two of the viruses are hypothesized to be multipartite (AbV6 and AbV16) on the basis of correlative abundance in different samples and a common 3′ motif.The 18 viruses have the closest amino acid homology to the RdRp of single-stranded RNA viruses. The assignment of ‘single-strandedness’ is at variance with both previous descriptions of the Sclerotinia sclerotiorum13 and between fungal species39. In this respect, the composition and abundance of A. bisporus viruses appears to be similar to that of plants and plant viruses, showing infection gains from the environment and losses probably due to competition and antagonistic interactions14. Local infection is inferred for sample 003 as both samples 003 and 004 were grown from A. bisporus inoculated compost from the same source, their fruitbodies collected on the same day, yet 003 had two additional viruses (AbV13 and AbV15) . Sequence variability ranged from 0.04–0.07 SNP’s/kb  suggesting a high fidelity replicase to two orders of magnitude higher for AbV16 RNA1, AbV14 and AbV16 RNA2 (8.9–10.1 SNP’s/kb) (Supplementary Table 5) Table . The mecAmbsetviridae proposed as a new viral family of plant and fungal viruses comprised the four component virus AbV16 and homologues found in the TSA and EST databases  and likely to be the aetiological agent of Brown Cap Mushroom Disease. The high variability found in AbV16 RNAs 1 and 2 is consistent with the high mutation rates associated with acute viruses35.The es Table . The comes Table . AbV16 m28, the 16.2 kbp RNA of Grogan et al.10 and the >13 kbp L-RNAs of Kuang et al.25; and ORFan2 is likely to be the 2.7 kb VX3 of Sonnenberg and Lavrijsen28, the2.4 kb S-RNA of Kuang et al.25 and the 2.4 kb RNA of Grogan et al.10 who identified this RNA in 99% of the 320 healthy or diseased mushroom samples tested. These ubiquitous and persistent viruses have clearly developed efficient strategies for replication35 which may have been amplified by cultivation and breeding of the host.The fruitbodies grown from a non-diseased commercial culture (sample 138) also harboured ubiquitous viruses and ORFans . Two of these show high similarity to those previously reported : the AbV2 sequence corresponded to the 14–17 kb RNAs of Sonnenberg and LavrijsenGeographical differences in the viral sequences were identified. The sequence of the North American ORFan2 . Three of the samples consisted of diseased mushrooms (displaying the fruitbody browning symptom) collected from different mushroom farms in 2004 and 2011  respectively.Fruitbodies were freeze dried and ground in liquid nitrogen. 3.5 g dry weight of each ground mushroom sample was suspended in 4x w/v of an STE-based lysis buffer, pH7, and RNA was extracted with an equal volume of 5:1 phenol chloroform (pH 4.5). The RNA was purified and enriched for dsRNA by the addition of ethanol to 16%, followed by chromatography through two cellulose columns , using a method modified from the protocol of Morris and DoddsThe purity of RNA extracts was assessed by absorption spectra using a Nano-drop 1000  and the level of degradation was assessed using size separation by capillary electrophoresis on a Fragment Analyser . Prior to sequencing RNA samples were quantified using the Qubit RNA BR Assay  and assessed for quality on a R6K Tapestation .cDNA and library construction were performed using NEBNext mRNA Sample Prep Master Mix Set 1  according to manufacturer specifications, with the following details: ~100 ng total RNA used per sample, no polyA or ribosomal depletion, bead size selection 200–600 bp and 12 cycles PCR. For samples 003, 004 and 1497, libraries were constructed using two different denaturation conditions (1) 72 °C for 3 mins or (2) 95 °C for 10 mins. For the remaining samples the denaturation conditions were 95 °C for 10 mins.Adapters were ligated using the Multiplexing Sample Preparation Oliogonucleotide Kit  and each library was size selected using Ampure XP beads . The following custom primers (25 µM each) were used for the PCR enrichment step: (1) Multiplex PCR primer; 1.0 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′, and (2) Index primer; 5′-CAAGCAGAAGACGGCATACGAGAT[INDEX*]CAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′, *The index refers to eight base tags developed by Wellcome Trust Centre for Human Genetics .The amplified library was purified using Ampure beads  and size distribution determined using a Tapestation D1K system . Libraries were quantified by Picogreen  and pooled to provide equal quantities of RNA per library.Finally, a Quantitative PCR was performed, using Agilent qPCR Library Quantification Kit and a MX3005P instrument , to measure the relative concentration of the pool compared to a previously sequenced mRNA library in order to determine the volume to use for sequencing. Sequencing was performed on an Illumina MiSeq with a read length of 150 bp, paired end reads and an average insert size of ~200 bp, at the Wellcome Trust Centre for Human Genetics .Agaricus bisporus genes, known rRNA, Pseudomonas and PhiX and these reads were excluded from the assembly.Fastqc was used to check the quality of the read data. Reads were trimmed using Trimmomatic to remove nucleotides with quality less than Phred33+15 and to only retain reads of 35 nucleotides or greater in length. Bowtie was used to align reads to De novo assembly was carried out using Velvet v1.2.08 and redundant contigs were removed using Oases v0.2.08 and the Fastx toolkit. Cap3 was used to assemble the smaller contigs into larger scaffolds, producing 39–140 contigs per sample.The NCBI Batch Web CD-Search Tool (default settings) was used to search the assembled contigs for conserved protein domains. Based on the domains returned contigs were assigned to one of four groups; (1) viral, (2) non-viral, (3) chimera  or (4) unknown (if no domains found). Contigs which were deemed to be of non-viral or chimeral origin were discarded.45 as well as the GC content for each of the contigs. The ORFs were interrogated using BLASTX to search for homology to viruses within the NCBI non-redundant protein sequence database. For sequences with no or low scoring BLASTX scores (e > 10−2), the translated ORFs were searched for viral homologies within the non-redundant protein sequence database using DELTA-BLAST. All contigs (and minor ORFs) which remained unidentified were further searched for homologies using HHpred46. The identified contigs were assigned numbers C1 to Cn.Open Reading Frames (ORFs) and the flanking Untranslated Regions (UTRs) were predicted using Geneious (6.0.6)All identified viral sequences were then searched for homology, using TBLASTX, to sequences within the Expressed Sequence Tag (EST) and Transcriptome Shotgun Assembly (TSA) databases, to identify further unidentified hypothetical viruses.47 implemented in Geneious (6.0.6)45, with iterations set to 100 and clustering method set to Neighbor-joining (other settings default). Bayesian inference trees where constructed using MrBayes (2.0.6)48 implemented in Geneious (6.0.6)45. Due to the divergent nature of some of the phylogenies, where indicated, Neighbor-joining trees were constructed using Geneious tree builder (6.0.6)45.Replicase sequences  for homologous and out-group viruses were downloaded from GenBank. Sequence alignment was carried out using MUSCLE34 was used to search for shared motifs in 5′ and 3′ UTR sequences. The maximum number of motifs to return was set to 20 and “Search given strand only” was checked. Other settings were kept as default. Pseudoknots were predicted in both 5′UTR and 3′ UTR using DotKnot_1.3.149.MEME 4.9.1To confirm the correct assembly of each contig, PCR primer pairs were designed to generate overlapping products of 600–900 bp in length designed to cover the entire length of the contig. The size of the PCR products was assessed using gel electrophoresis and any anomalous products were Sanger sequenced by GATC Biotech .The 5′ end of each contig was determined using 5′ RACE PCR , using the manufacturer’s protocol, followed by Sanger sequencing of the product by GATC Biotech .50. Reads were summed and genotype likelihoods calculated with samtools v1.3.1 mpileup51. The reference sequence for each virus was defined as the most abundant sequence present in sample 003 where possible and sample 2990 for the remaining RNAs. The variant positions were called with bcftools v1.3.151, counting only variants present when the total read depth in the region was greater than 10. The number of SNPs present in each contig was summed for each sample and then the mean number of SNPs per contig was calculated.Contig sequence variability was assessed by determining the number of SNPs per kb of sequence. The reads for each sample were aligned to the reference viruses separately using Bowtie2 v 2.0.5Supplementary Information


HTT sequences in fibroblasts derived from patients. We tested reagents that either acted as translation blockers or triggered mRNA degradation via the RNA interference pathway or RNase H activation. We also analyzed the effect of chemical modifications of CAG repeat-targeting siRNAs on their efficiency in the foci decline. Our results suggest that the decrease of RNA foci number may be considered as a readout of treatment outcomes for oligonucleotide reagents.In several human polyglutamine diseases caused by expansions of CAG repeats in the coding sequence of single genes, mutant transcripts are detained in nuclear RNA foci. In polyglutamine disorders, unlike other repeat-associated diseases, both RNA and proteins exert pathogenic effects; therefore, decreases of both RNA and protein toxicity need to be addressed in proposed treatments. A variety of oligonucleotide-based therapeutic approaches have been developed for polyglutamine diseases, but concomitant assays for RNA foci reduction are lacking. Here, we show that various types of oligonucleotide-based reagents affect RNA foci number in Huntington’s disease cells. We analyzed the effects of reagents targeting either CAG repeat tracts or specific  HTT gene. Both RNA and protein products from the mutant allele are proposed to be involved in the pathogenic process; therefore, the most promising therapeutic approaches are designed to target the mutant transcripts. Mutant mRNA is partially or temporarily retained in the nucleus within splicing speckles, as shown in multiple types of cellular models of polyQ diseases , myotonic dystrophy type 2, fragile X–associated tremor/ataxia syndrome, and amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD)  non-functional reagents that neither affect the size, number, and morphology of foci nor protein or RNA levels; (II) reagents that decrease RNA and protein levels, but do not affect RNA foci, indicating the degradation of RNA only in the cytoplasm; (III) reagents that decrease only protein levels, acting as translation blockers; (IV) reagents that decrease RNA foci as well as protein and RNA levels, indicating the degradation of transcripts in both the nucleus and cytoplasm; (V) reagents that decrease the foci number, but increase RNA and protein levels, suggesting the release of transcripts from foci to the cytoplasm, without further degradation; and (VI) reagents that increase the number of RNA foci by triggering the retention of RNA in the cell nucleus.HTT gene, HD2: GM09197, 21/151 CAG in HTT gene) were obtained from Coriell Repository . Fibroblasts were cultured in Minimal Essential Medium  with 10% Fetal Bovine Serum (Sigma–Aldrich) supplemented with GlutaMAX , non-essential amino acids (Sigma–Aldrich), and Antibiotic Antimycotic Solution (Sigma–Aldrich) at 37°C. All cell cultures were checked for mycoplasma contamination.Human fibroblasts  or IDT , and duplexes were annealed according to the manufacturer’s instructions. The LNA oligomer was synthetized by Exiqon . The sequences of the synthetic ONs and oligomer used in this study are presented in Cell transfections were performed using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. The reagents were transfected at 50 nM, unless stated otherwise in figure legends. Oligonucleotide treatment lasted for 4 h; after that time medium was changed. Material for subsequent analyses was collected after 48 h. For fixation, cells were seeded directly on cover slips prior to transfection. The transfection efficiency was monitored using BlockIT fluorescent siRNA (Life Technologies).in situ hybridization and IF procedures were performed as described previously (2 Fragment Donkey Anti-Mouse IgG (H+L) antibody labeled with Alexa488 . For RNase treatment, cells were subjected to 0.01 μg/ml RNase A for 1.5 h after the permeabilization step. For IF, samples were blocked with 1% BSA buffer for 1 h at RT, incubated with primary anti-huntingtin antibody  at 4°C overnight and with secondary antibody AffiniPure F(ab′)2 Fragment Donkey Anti-Mouse IgG (H+L) antibody labeled with Alexa488 (1:500) for 1 h. For combined RNA FISH and IF, first, FISH was performed with only 1x SSC washings. Next, samples were blocked with 1% BSA for 30 min and incubated with anti-huntingtin antibody for 3 h and secondary antibody for 45 min. For endosomes visualization anti-Rab5  and anti-EEA1  antibodies and secondary antibody AffiniPure F(ab′)2 Fragment Donkey Anti-Rabbit IgG (H+L) antibody labeled with Alexa488  were used. Nuclei were imaged with SlowFade Gold with DAPI (Life Technologies). Images were captured with a Leica SP5 confocal microscope.Fluorescence eviously . Briefly6CA , for foci imaging we used either LNA-modified DNA probe Cy3-(CTG)6CT  or three HTT-specific 5′-Biotin-labeled DNA probes: GGTAAAAGCAGAACCTGAGCGGCCGTCCATCTTGGACCCGT, TCGAAGGCCTTCATCAGCTTTTCCAGGGTCGCCAT, TCCATAGCGATGCCCAGAAGTTTCTGAAATTCTGGAG (Metabion).For CAG-specific ONs detection in cells we used DNA probe Cy3-(CAG)The western blot analysis for the HTT protein (17/68Q tract) was performed as previously described . BrieflyHTT, GAPDH, or U6-specific primers  and a Direct-zol Kit  according to the manufacturer’s instructions. For isolation of RNA fractions Cytoplasmic and Nuclear RNA Purification kit  was used according to the manufacturer’s instructions. The RNA concentration was measured using a DeNovix spectrophotometer . An amount of 500 ng of total RNA or 200 ng of fractionated RNA was reverse transcribed at 55°C using Superscript III (Life Technologies) and random hexamer primers . cDNA was used for qPCR using LightCycler 480 SYBR Green I Master  with denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and elongation at 72°C for 20 s, with ImageJ software (Fiji distribution) was used for image analysis. Images were adjusted with respect to brightness, contrast, and smooth effects. For the statistical analysis of images, a Python script for ImageJ was prepared.For estimation of RNA foci number Python (Jython) script was prepared for ImageJ. First, analyzed area was restricted to the nucleus using DAPI signal. Next, after adjusting threshold for the red signal (from probe), image was converted to mask and particles were analyzed . Each image represented single cell. Results for group of images were saved in text file for statistical analyses.For estimation of quantity of nuclear CAG transcripts Python (Jython) script was prepared for ImageJ. First, analyzed area was restricted to the nucleus using DAPI signal. Next, mean intensity signal  from the nucleus was calculated. Each image represented single cell. Results for group of images were saved in text file for statistical analyses.t-test, with an arbitrary value of 1 assigned to cells treated with control siRNA (siLUC). Selected data were compared using an unpaired t-test with Welch’s correction to assess the allele-selectivity of silencing . The statistical significance of the number of foci, nuclear RNA signal and percent of aggregate-positive cells was assessed using a one-way ANOVA and multiple comparisons testing with post hoc Dunnett’s tests. For FISH and IF analyses, at least 30 cells were analyzed for each experimental condition. p-values of <0.05 were considered significant.All experiments were repeated at least three times. The statistical significance of changes in gene expression levels  was assessed using a one-sample HTT sequence or CAG repeats), (II) anticipated mechanism of activity , and (III) pattern of chemical modification (pure RNA ONs and RNAs containing 2′-fluoro (2′F), 2′-O-methylo (2′OMe), and phosphorothioate (PTO) modifications, DNA gapmers with 2′OMe and PTO, as well as LNA oligomer). All ON sequences are presented in Table 1 with references if they were used in previous studies. Specifically, we used siRNA and ASO targeting HTT-specific sequences  and a set of CAG repeat-targeting ONs: unmodified RNA duplex (CAG/CUG), miRNA-like siRNAs with single mismatches (A2 and G2), chemically modified siRNA (A2F and A2M), antisense oligonucleotide (ASO CTG) and a short LNA blocker (LNA CTG) .We used a set of chemically synthesized ONs that differed with regard to their (I) targeted sequence . We observed predominantly cytoplasmic localization of RNA interference reagents, both unmodified and chemically modified. Owing to the differences in binding strengths of the probes to various ONs, we did not quantify differences between reagents. The ONs mainly localized to cytoplasmic vesicles , similar to the localization observed for control fluorescent siRNA, BlockIT (data not shown). Observed ONs did not localize within endosomes marked with EEA1 and Rab5 proteins showing that these are probably vesicles formed by the lipofection . ASO localized within both the cytoplasm and nucleoplasm; however, a strong bias toward the nucleus was observed. Non-transfected cells and cells transfected with control siRNA siLUC were used as negative controls. Next, we examined whether the CAG repeat-expanded tract present in HD cells affects the localization of reagents targeting the CAG sequence. We did not observe significant differences in localization between HD and control fibroblasts, indicating that the presence of the mutation in cells does not alter reagent localization .First, we aimed to determine the cellular distribution of ONs targeting CAG repeat sequences. Selected siRNAs , ASO CTG, and LNA CTG were delivered by lipid-based transfection. After 48 h, cells were fixed and small-RNA FISH was performed using probes that interact with the ONs in a 1:1 stoichiometry. However, we were not able to visualize LNA reagents owing to their short length. First, we obtained images of reagents in control fibroblasts . Both analyzed HD lines demonstrated nuclear RNA foci, which were observed in about 50% cells. RNA foci were not detected after RNase treatment . An additional feature of HD that may serve as an indicator of an effective therapeutic approach is the presence of HTT protein aggregates. We observed huntingtin aggregates in the cytoplasm and nucleus in both HD fibroblast cell lines, and no such aggregates were visible in the control cell line . Thus in addition to western blotting, IF may be used to observe protein level changes resulting from ON treatment.In our earlier studies we defined the effectiveness of ON treatment at the RNA level using RT-PCR  and at the protein level using western blot , 2016. THTT expression at the mRNA and protein levels using selected ONs under the same experimental conditions as used for microscopic analyses, i.e., 48 h after the transfection of HD fibroblasts with 50 nM ONs. We assessed total HTT mRNA levels using qRT-PCR and primers located downstream the CAG repeat tract . The most significant decreases in HTT mRNA were observed for siHTT (to ∼40% of the control level) and for both ASOs (to ∼65% of the control level). CAG/CUG reagent decreased level of HTT mRNA to ∼85%. A separate analysis of HTT alleles by semi-quantitative RT-PCR revealed a similar general trend of HTT silencing by these ONs and there was no indication of allele-selective inhibition by ASO reagents (data not shown). At the selected time point, we did not observe decrease in HTT mRNA level by A2, G2, A2F, and A2M ONs. Additionally, for selected set of ONs we performed RNA fractionation to analyze changes in nuclear and cytoplasmic transcript level  but more prominent effects were observed in the cytoplasm what in total resulted in HTT mRNA decrease to ∼25%. For A2 we observed slight increase of HTT transcript in the nucleus and slight decrease in the cytoplasm. As expected, the effects of ASOs activity were mainly nuclear as these ONs decreased HTT mRNA level to ∼45% already in the nucleus. For all nine ONs we performed western blotting to analyze normal and mutant huntingtin levels separately . For ONs that lowered HTT mRNA (siHTT and ASOs), significant decreases in protein levels were also observed, to ∼25%, ∼30%, and ∼60% of the control level for siHTT, ASO HTT and ASO CTG, respectively. Neither siHTT, nor any of the used ASOs acted in an allele-selective way, decreasing both alleles with similar strength. Consistent with previous reports, selected CAG repeat-targeting reagents caused the allele-selective lowering of huntingtin, with a high preference for the mutant protein observed for A2, G2, and A2F. Significant allele-selectivity was also observed for A2M ON, but not for CAG/CUG siRNA or LNA CTG.We investigated the silencing of Figures 4A,B). Untreated HD fibroblasts had a mean of 12.4 foci per foci-positive cell, in agreement with our previous reports. Cells treated with siLUC and BlockIT exhibited similar means, with 11.2 and 14.3 foci per cell, respectively. Most significantly RNA foci-disrupting ONs were A2, with a mean of 6.6 foci per cell, ASO CTG, with a mean of 6.1, and LNA CTG, with a mean of 6.8 foci per cell . Most of the tested ONs significantly decreased the number of foci per cell, but with different efficiencies. siHTT, CAG/CUG, G2, A2F, and A2M reduced foci number to a mean of 7.9, 7.4, 7.3, 7.5, and 8.1 foci per cell, respectively. We did not observe significant foci alterations using ASO HTT. Next, we analyzed nuclear signals from the CTG probe . All HTT-specific and CAG-specific ONs, except for LNA CTG, decreased level of detected RNA in the nucleus. siHTT and CAG/CUG reagents also triggered visible reductions in overall signal intensity from the cell. Differences between results obtained for measurement of nuclear level of transcript with RNA FISH and qRT-PCR after fractionation . There was no clear correlation between the reduction in RNA levels and the RNA foci phenotype .Next, we analyzed RNA foci after ONs treatment to investigate whether mRNAs within the nucleus are accessible to ON reagents, whether these ONs exert their activity within the nucleus, and whether they can decrease RNA foci. Our results showed that not all tested ONs are able to significantly decrease the number of RNA foci, despite altering the RNA or protein levels. The results differed for probes used; however, a similar tendency to reduce number of RNA foci was observed . However, we could additionally observe differences in protein localization after ON treatment and the presence and number of mutant protein aggregates. Protein aggregates were observed in cells with or without RNA foci showing that there is no connection between formation of RNA foci and protein aggregates . Moreover, in a substantial portion of cells, the protein localized around the nucleus after ON treatment . Next, we measured the percentage of aggregate-positive cells . With the decrease in mutant protein levels observed by western blotting, we also observed decreases in the number of protein aggregates. In untreated and siLUC-treated fibroblasts, we observed 1–3 aggregates in about 60% of cells. Protein aggregates localized mainly in the cytoplasm. Rarely, cells with a high number of cytoplasmic protein aggregates were observed and these cells also had nuclear aggregates . We observed significant decrease in aggregate-positive cells up to 35–38% after treatment with A2M, G2, and A2F .To further analyze the therapeutic potential of selected ONs, we performed IF experiments to detect huntingtin protein  in cells. The results of the microscopic analysis of HTT protein levels (Molecular processes involved in the manifestation and progression of polyQ diseases are not fully known; however, in addition to the roles of mutant proteins, the roles of mutant RNA in the pathomechanism of these diseases are increasingly recognized. RNA toxicity mechanisms include the formation of nuclear foci, protein sequestration within these foci triggering alternative splicing events and gene expression defects as well as aberrant biogenesis of small CAG-repeated RNAs . AccordiHTT mRNA can be targeted by ON-based reagents in the nucleus, as the decrease in number of RNA foci was observed. This may be explained by the dynamic nature of foci, but may also indicate that the ONs examined in this study were incorporated into nuclear speckles or, alternatively, targeted mutant transcripts preventing their detention in nuclear speckles. The factors that distinguish the reagents and determine their potential to reduce RNA foci may be their cellular localization and mechanism of their action (Figure 6 and Supplementary Table in vivo. LNA CTG treatment led to rescue of lowered levels of striatal markers and improved motor functions in HD mouse model. It was also shown that used ON was able to decrease number of foci-positive cells (As mutant RNA foci are found in the nucleus, it was not clear whether the ONs used would be able to affect RNA foci in any way. According to recent studies, RNA foci are not static, but rather dynamic structures, as shown for CGG and CUG repeats , and mutve cells . LNA CTGHTT mRNA levels. Using siHTT, we showed that despite a strong decrease in transcript levels, the influence on RNA foci was less than the effect observed for other tested ONs. These findings are in agreement with earlier reports indicating that siRNA against C9orf72 RNA in ALS does not significantly alter RNA foci, despite a significant decrease of C9orf72 RNA levels (C9orf72 RNA, both repeats and specific sequence, led to decreases in the number of cells containing foci as well as the number of foci per single cell (The comprehensive approach used in this study to compare various ONs in a single experimental model and conditions enabled us to draw general conclusions about the decrease in RNA foci phenotype. First, we observed that decreases in RNA foci number are not directly correlated with decreases in A levels . On the gle cell . HoweverOur results do not answer the question whether the observed reduction in foci number results mainly from the inhibition of new foci formation by ON binding to expanded CAG repeats in nucleoplasm or from deconstruction of already existing foci formed by mutant transcript which is detained in nuclear speckles. The presented results demonstrate that the ONs bind to mutant transcript already in cell nucleus and we hypothesize that this binding altering accessibility of CAG repeats in mutant transcript prevents its interaction with unidentified yet factor responsible for transcript nuclear detention.WK, MU, and AF designed the study. MU performed FISH/IF and all microscopic analyses using ImageJ and prepared the figures and tables. AF performed western blotting, fractionation and real-time PCR experiments, including data analysis. MU and WK wrote the paper.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.


With combination of multiple techniques, we have successfully characterized unique, complex chromosomal changes in a patient with chronic lymphocytic leukemia (CLL), a lymphoproliferative disorder.The diagnosis was based on white blood cell, flow cytometry, and immunophenotypes and confirmed by karyotype, fluorescence in situ hybridization, and array comparative genomic hybridization from the patient's blood culture.The patient was given fludarabine, cyclophosphamide and rituximab (FCR) for 6 cycles.After completion of 6 cycles of FCR, the computed tomography scans of the neck/chest/abdomen/pelvic showed that the patient in CR. During the 10-month follow-up, the patient's clinical course remained uneventful.The translocation t identified in this patient is a recurrent translocation found in patients with chronic B-cell lymphoproliferative disorders and the 3-way translocation involving chromosomes 2, 14, and 11 may play a role as an enhancer. In addition, del17p13 involving deletion of the tumor suppressor gene TP53 occurs in <10% of CLL at diagnosis but up to 30% in refractory cases. Patients with del17p demonstrate aggressive diseases and have very poor prognoses.–7B-cell chronic lymphocytic leukemia (CLL) is a genetically heterogeneous neoplasm characterized by the progressive accumulation of CD5+ mature B cells in bone marrow, lymph nodes, and blood. CLL is the most common leukemia in adults in Western countries and the clinical course of disease ranges from a few months of the diagnosis to ≥20 years.IGH) rearrangement on 14q32 are relatively infrequent in CLL with a frequency of 4%. However, recent studies have revealed that translocations involving IGH rearrangements occur at a very high incidence rate and significantly affects survival of CLL patients.–10 The most frequent translocation in CLL is t , which juxtaposes IGH and BCL3 resulting in overexpression of BCL3 and is usually associated with unfavorable clinical outcome and trisomy 12.,12 It was proposed that these 2 changes might cooperate for malignant transformation. In contrast, patients with del(13q) have a better survival than the patients with other cytogenetic abnormalities. Furthermore, t is also a recurrent chromosomal change in CLL.It has been reported that chromosomal translocations involving immunoglobulin heavy chain locus  to generate FGFR3/IGH, t to form CCND1/IGH, t to produce IGH/BCL2 fusion, and t have been identified and provided further insights into the pathogenesis of CLL.–15 However, complex variant translocations may occur in CLL but have been rarely reported.Chromosomal translocations are regarded as an important prognostic indicator and are always associated with shorter survival in B-CLL patients.In the present study, as confirmed by cytogenetic analysis, we report a patient carrying the classical t as well as a novel 3-way translocation t, trisomy 12, and del(13q14). Importantly, we revealed a cryptic gain of chromosome 4p16.2 besides trisomy 12 and del(13q14.11-q21) in this patient. This study was approved by the institutional review board (IRB) at the University of Oklahoma Health Sciences Center .22.13 cells/μL (140–440 × 103 cells/μL), respectively. Her white blood cell count was 21.81 × 103 cells/μL (4–11 × 103 cells/μL) with relative and absolute lymphocytosis of 67% (15%–46%) and 15.7 × 103 cells/μL (0.6–5.1 × 103 cells/μL), respectively. Flow cytometric analysis found that her 75% monoclonal B-cells showed lambda light chain restriction of moderate intensity; her immunophenotype were as follows: CD5+, CD10-, CD19+, CD20+, CD22(dim), CD23 +/− (dim), FMC-7 +/− (dim), CD38+.A 43-year-old female was admitted to the University of Oklahoma Health Sciences Center where she was diagnosed with CLL owing to weight loss and lymphadenopathy. Her hemoglobin and platelet counts were 12.8 g/dL  and 158 × 102.2At diagnosis, the patient's B-cells were subjected to karyotype analyses. The results revealed that 25% (5/20) of the metaphase chromosome displayed a variant translocation among chromosomes 2, 11, and 14, and a translocation between 2 chromosomes 14 and 19 as well as +12 and del(13)(q14.11-q21). The karyotype was designated as 47, XX, t , +12, del(13)(q14.11q21), t   and LSI BCL11A and LSI BCL3 dual color break-apart rearrangement probes . The uncultured cells were also tested using CLL panel . All the experimental procedures followed the manufacturers’ instructions.Fluorescence in situ hybridization (FISH) analyses were performed in the uncultured and cultured cells using the MLL gene break-apart signals in 16% of 200 examined cells were observed as demonstrated by 1 tiny Spectrum Green signal and 1 normal Spectrum Red signal in Figure MLL gene break-apart. Furthermore, 104 of 200 cells (52%) exhibited a biallelic rearrangement of IGH (14q32) as showed by Spectrum Red and Spectrum Green signals when compared to Spectrum Orange signals in the normal IGH q13;q32), t, del(6)(q23), del(11)(q22), del(17)(p13), but found trisomy 12 in 74% and monoallelic 13q14 deletion in 21% of tested cells. Moreover, variant , t and 5’IGH were translocated to der(11) and der(19), respectively  and 3’BCL3 was translocated to der(14)  and 3’BCL11A was translocated to der(14)  and 3’MLL was translocated to der(2)  gene  analyses on the patient's DNA sample revealed the presence of an extra chromosome 12 and deletion of 13q14.11-q21, which was consistent with our karyotype analyses. Interestingly, we also found a gain of 4p16.2  containing 3IGH;BCL3 and t /BCL11A;IGH;MLL were observed simultaneously in one patient in addition to trisomy 12 and del(13q). In addition, we detected a microduplication in 4p16.2 involving the MSX1 gene that plays an important role during early hematopoiesis. Importantly, we found a biallelic rearrangement of IGH (14q32) with partner genes BCL11A, MLL, and BCL3 in our case.The case reported here is unique and particularly interesting, as 2 balanced translocations including t/IGH not only promote pathogenesis but also predict a poor prognosis of B-cell malignancies such as t(MYC/IGH) in Burkitt lymphoma (BL); t (CCND1/IGH) in mantle cell lymphoma (MCL); and t (IGH/BCL2) in follicular lymphoma (FL). The cases with concomitant t and t translocations have previously been reported, and they are always regarded as a rare recurrent chromosomal changes associated with atypical cytology, trisomy 12, and a progressive disease in CLL.,17,18Translocations involving BCL11A;IGH;MLL, especially concurrent with t/IGH;BCL3. In contrast to MYC/IGH, CCND1/IGH and IGH/BCL2, the roles for IGH;BCL3 and BCL11A;IGH;MLL fusions in CLL remain poorly understood; however, it is possible the target genes that become overexpressed or gained new functions may be relevant to the poor prognosis of CLL.The present study is the first report of a CLL case with a complex variant translocation involving 3 chromosomes 2, 11, 14 named t/MLL gene rearrangement often occurs in acute myelocytic leukemia (AML), acute lymphoblastic leukemia, and myelodysplastic syndrome. In hematologic malignancies such as CLL, the most common abnormality is the deletion of the MLL(11q23), whereas the MLL gene rearrangement has not been previously observed in CLL. In this study, we revealed this interesting 3-way translocation of the MLL gene rearrangement; whether it contributes to the leukemia progression or even an unfavorable prognosis in CLL warrants further investigation.The IGH rearrangement is not sufficient to induce tumorigenesis, and acquisition of additional genetic aberrations is necessary for malignant transformation. Trisomy 12, observed in this case, is one such genetic anomaly. The cytogenetic abnormality of trisomy 12 associated with intermediate prognosis is observed in up to 50% of IGH/BCL3-positive B-CLLs and was considered to act cooperatively with t in leukemogenesis. Interestingly, however, it was reported that patients with 13q deletions as a sole abnormality had the longest estimated survival times compared with other cytogenetic abnormalities.,21 Moreover, miR-15a and miR-16–1 locate in this region, and negatively regulate BCL2 expression at a posttranscriptional level.It is widely believed that presence of only the MSX1 was found to be overexpressed in cell lines derived from MCL and leukemia AML as well as in 3% of patients with MCL and AML. In the present study, array CGH revealed a cryptic gain of MSX1 gene besides trisomy 12 and del(13q14.11-q21), which has not been reported previously in CLL. These data suggest an oncogenic role for MSX1 in leukemogenesis.IGH/BCL3 translocation and a three-way variant translocation BCL11A/IGH/MLL, as well as trisomy 12 and del(13q). Furthermore, a cryptic genomic alteration involving leukemia-related MSX1 gene was found in this case at the level of the array CGH.In summary, we reported a rare case of an adult CLL patient with the coexistence of classical


Caenorhabditis elegans. We found that mechanical stress induces supercoils and plectonemes in the sensory axons of spectrin and tau double mutants. Biophysical measurements, super-resolution, and electron microscopy, as well as numerical simulations of neurons as discrete, elastic rods provide evidence that a balance of torque, tension, and elasticity stabilizes neurons against mechanical deformation. We conclude that the spectrin and microtubule cytoskeletons work in combination to protect axons and dendrites from mechanical stress and propose that defects in β-spectrin and tau may sensitize neurons to damage.Our bodies are in constant motion and so are the neurons that invade each tissue. Motion-induced neuron deformation and damage are associated with several neurodegenerative conditions. Here, we investigated the question of how the neuronal cytoskeleton protects axons and dendrites from mechanical stress, exploiting mutations in UNC-70 β-spectrin, PTL-1 tau/MAP2-like and MEC-7 β-tubulin proteins in DOI:http://dx.doi.org/10.7554/eLife.20172.001 Neurons play a central role in receiving and distributing information and depend on long, slender axons and dendrites for their function. Vertebrate peripheral neurons can be meters long and less than 1 μm in diameter: the peripheral neurons of Cetaceans have aspect ratios exceeding a factor of 10,000,000 . DespiteAxons harbor bundles of microtubules (MT), which are known to be important for neuronal architecture, growth, and organelle transport . MTs areC. elegans neurons to buckle and deform during body movements, leading to axon damage  required for sensing gentle touch  that fill the cytoplasm. To understand how these structures interact to protect axons and dendrites from mechanical stress in living animals, we examined axon shape in mutants carrying genetic defects in the constituents of actin-spectrin networks and MT bundles. Our study builds on prior work in this area: Genetic defects in UNC-70 β-spectrin have been linked to movement-dependent axon fracture  and bucknsferase  and miss-tubulin . No such-tubulin , suggestunc-70(e524) missense mutants. The e524 allele encodes a missense mutation, E2008K, affecting the linker region between the tetramerization domain and the 16th spectrin repeat of β-spectrin . Using this approach, we examined a novel allele of mec-7 β-tubulin, wy116, and a null allele of ptl-1 tau, ok621.To learn more about the interaction between the spectrin and the microtubule cytoskeleton, we analyzed TRN shape in mutants affecting the microtubule cytoskeleton and asked whether or not these mutations could enhance shape defects in spectrin . To enabwy116 allele replaces threonine 409 with an isoleucine  and has a mild defect in touch sensation (mec-7(wy116) animals were straight and lacked local bends and shape distortions (mec-7(wy116);unc-70(e524) double mutants displayed large bends and kinks that were not found in either single mutant. Such shape defects were largely absent in mec-12(e1607); unc-70(e524) double mutants (wy116 affects a region implicated in binding MT-associated proteins (MAPs) including tau (C. elegans genome (The ensation . As in wtortions . The TRN mutants  that lac mutants . This fiding tau  led us ts genome .10.7554/mec-7(wy116), TRNs in ptl-1(ok621) null mutants were straight, lacked obvious shape distortions, and had D values close to zero (ptl-1(ok621);unc-70(e524) double mutants, by contrast, the TRN axons exhibited large bends and localized helices, loops, and plectonemes (ptl-1(ok621);unc-70(e524) double mutants expressing wild-type UNC-70 β-spectrin in the epidermis from the oxIs95[epi::UNC-70] transgene. This manipulation resulted in a less pronounced phenotype but did not obviously alter the displacement metric, D  along the axon ;unc-70 double mutants  and the ALM axons lie much closer to the neutral bending axis at z = 5.5 ± 1.7 µm (n = 15 worms). If Euler’s simple theory of mechanical stress in a bending cylindrical beam holds for C. elegans, then the body bends that occur during movement are expected to deform the AVM axon four-fold more than the ALM axon.Mechanical stresses generated by tissue movement are not expected to be uniform throughout the tissue. The worm’s cylindrical body and the differential position of the lateral  and ventral  TRNs provide an opportunity to investigate this question. Given that ing axis . Euler bing axis  predicts animals , the AVMhim-4 hemicentin that displaces the ALM away from the neutral bending axis and toward the dorsal side of the animal B=EI than those treated with the actin depolymerizing agent Latrunculin A  β-spectrin mutant axons  were lower than untreated wild-type axons and indistinguishable from Latrunculin A-treated wild-type axons (p=0.28). We repeated these measurements and compared wild-type axons  to those of ptl-1(ok621)  and mec-7(wy116) neurons  and found that both classes of mutant axons had significantly lower Young’s moduli, in agreement with our hypothesis that mutations affecting the microtubule cytoskeleton lead to decreases in bending rigidity. Collectively, these results demonstrate that axonal stiffness depends on both the actin-spectrin cytoskeleton and MT bundles.To obtain initial values for the relevant mechanical parameters, we estimated the Young’s modulus from elasticity measurements of cultured, GFP-tagged TRNs by indenting them with an atomic force microscope  was not sufficient to induce the formation of coils or plectonemes for 10-fold variations around the experimentally determined input moduli . An incrτcrit . The anafness, B , axial sss, B τcritkcrit, which is inversely proportional to B:For torques that exceed τ and C.This indicates the boundary  of MTs yields a bending stiffness of 1.26 In the classical description of rods, the mechanical parameters  area, A . Howeverals e.g. . Modelin10−23N m . This yi10−23N m  and the 10−23N m . Similar10−23N m .mec-7(wy116);unc-70 and ptl-1;unc-70 double mutants (mec-17 (mec-12(gm379)   might arC. elegans TRNs in vitro , suggesting that the spectrin tetramer resides at its fully extended contour length in these cells.To examine the architecture of actin-spectrin networks, we used super-resolution microscopy  to visualize SPC-1 α-spectrin and UNC-70 β-spectrin in transgenic animals carrying fluorescent proteins fused to their C- and N-termini, respectively . In agrein vitro  with an unc-70(e524) mutants  neurons is consistent with the decreased axial tension found in these mutants in vivo  TRNs, by contrast, the distribution of edge-to-edge distances was altered such that the geometric mean decreased to 4 nm, or about half the value in wild-type (2 = 32 and 89 nm2 in wild-type and mec-7(wy116) respectively). Despite this, a hexagonal arrangement of MTs was also detected in mec-7(wy116) axons , although the 120° and 180° peaks seemed collapsed together in the histograms. However, individual MTs often separated from the main bundle and adjacent MTs often contacted one another in these mutants (mec-7(wy116); unc-70(e524) double mutants  axons  with sim mutants , situati mutants . Qualita mutants . This va mutants .10.7554/mec-7(wy116) missense mutants, MTs were closer together in ptl-1(ok621) null mutants than in wild-type axons ;unc-70(e524) double mutants (ptl-1(ok621) single mutant ;unc-70 mutants consistent with having sectioned a local coil within the TRN axon  and ptl-1(ok621) mutants exhibit similar defects in MT bundle structure, we hypothesized that wy116 disrupted the association between the PTL-1 protein and microtubules. To test this idea in living animals, we used CRISPR/Cas9-based genome editing , which agrees with values reported for mammalian tau in vitro  β-tubulin null mutants and in mec-7(wy116) β-tubulin missense mutants. By comparison, PTL-1 mobility was unaffected in unc-70(e524) mutants (mec-7(wy116) could reflect a decrease in the number of MTs  had no detectable effect on PTL-1 mobility  mutants, which retain a periodic spectrin cytoskeleton, and unc-70(s1502) mutants, in which the spectrin network is disrupted  mutant ALM neurons than that in either wild-type (F-test p=0.01) or unc-70(e524) ;oxIs95[epi::UNC-70] and unc-70(s1502);oxIs95[epi::UNC-70] animals in which wild-type UNC-70 β-spectrin was expressed in the epidermal cells that engulf the ALM neurons. We found that MT length and number in unc-70(e542);oxIs95[epi::UNC-70] transgenic animals were similar to those of unc-70(e542) mutants (unc-70(s1502) mutants (unc-70(e524) animals acts in a dominant fashion and interferes with the mechanical stabilization that would otherwise result from the expression of wild-type UNC-70 β-spectrin in the epidermis.To further examine the possibility that the shorter MTs in the β-spectrin mutants might result from mechanically enhanced fracture and/or disassembly, we generated  mutants . However mutants . One plaTogether, these findings suggest that the longer ALM MTs are more susceptible to bending-induced fracture than the shorter AVM MTs. In addition, an analysis of MT length and number in β-spectrin mutants suggests that the actin-spectrin network provides a lateral support that protects the MT bundle against deformation via both cell-autonomous and non-autonomous mechanical stabilization.ptl-1 single mutants and ptl-1;unc-70 double mutants  triple mutant. e1434 is a temperature-sensitive, semi-dominant, missense allele that encodes a P171I mutation in MEC-7 β-tubulin  triple mutants were cultivated at a permissive temperature (15°C) that allows for nearly wild-type MT assembly  under compressive forces in cultured hippocampal neurons ] to express wild-type UNC-70 β-spectrin in the epidermis (pgIs22 [unc-70p:: UNC-70(N-TSMod)] to mark the N-terminus of UNC-70 , we used a self-excising cassette variant of CRISPR/Cas9 genome editing , GN119 uIs31 ptl-1(ok621), GN115 uIs31; mec-7(wy116), and GN643 uIs31 ptl-1(ok621);unc-70(e524) animals. TRNs were identified in these mixed cultures by their expression of GFP. Force-indentation (F-d) curves were taken at 7 μm−1 with a contact force of 500 pN. The contact geometry was modeled as a Hertzian impact between an incompressible sphere indenting the axon as a cylinder ; K1 = complete elliptic integral of the first kind ~1.71; K2 = complete elliptic integral of the second kind ~0.89; d = diameter of the tip ~60−9 nm; K1 and K2 are from Case 11: Sphere in contact with a cylinder  in Ds = 60 nm.Using the following values: We used a commercial STED instrument mounted on a Leica SP8 confocal microscope to analyze the subcellular organization of native actin-spectrin networks. We excited fluorophores on anti-GFP antibodies with a pulsed 80 MHz white light laser at 488 nm and depleted them with a continuous 592 nm laser through a 100x/1.4 oil immersion objective lens. The depletion laser power was adjusted between 30 and 40% of its maximum output power. A time gate was used to reject photons with a lifetime outside a 1.3 and 6 ns time window. To achieve optimal sampling at the Nyquist frequency of the expected resolution (~50–70 nm), the pixel size was matched to be ~20 nm. The scan rate was set to 100 Hz, while averaging each line 8–16 times. All STED imagings were performed on fixed samples of cultured neurons  axis of the neurite with a second random component along the dorsal-ventral axis. Through this approach, we derived a measure of the degree of deviation from a straight path from the mean-squared displacement (MSD) in the dorsal-ventral axis, We analyzed neuron shape in living animals as follows. First, we imaged young adult animals carrying the  in Fiji , binarizx is the displacement along the anterior-posterior or long axis and y is the displacement along the orthogonal dorsal-ventral axis, N is the number of samples (pixels or contour length encompasing the neuron), and n is the rolling window size (e.g. in pixels or segmentlength). For each segment length n, we sum over all possible windows that fit into the contour of the neuron and normalize by the number of 'measurements' (or windows/segments). For a straight line, the error will be zero and the MSD will be the square of the contour.Here, n and fit the curve to expected relationship:Next, we plotted MSD versus S is the persistence and D is used as a measure of the deviation of the neuron from a perfectly straight path In Experimental: We used a confocal microscope (Leica SP8) to perform FRAP experiments on ptl-1(pg73[PTL-1::mNeonGreen]) III. A small region-of-interest was bleached by scanning the area at 100% power with the 488 nm line of an Argon ion laser. Prior to and following the bleaching phase, power was attenuated to 0.5–2%. Three images were acquired at 1.428 Hz prior to bleaching and the recovery from photobleaching was monitored at 1.428 Hz for 10 frames and 0.2 Hz thereafter. This strategy enabled us to capture the initial and rapid recovery from bleaching.Theory: The recovery process was modeled as diffusive process in one dimension, with an infinite reservoir of emitters (fluorophores) inside the axon. To analyze the recovery process, we follow the approach outlined by C as the concentration of the emitters (fluorophores), t as the time and x as the direction of the diffusing species (corresponding to the orientation of the axon) and D as the diffusion constant. The solution has a Gaussian shape: M the total amount of the diffusing species, which is, in the case of an FRAP experiment, the bleached emitters diffusing out of the bleach spot. It is readily apparent that the dispersion of the Gaussian profile, w(t), is related to the diffusion constant according to 2 was a function of time for a series of intensity profiles obtained from images taken during the recovery process and straight line with slope 2D passing through the origin is expected. Similarly, the change in amplitude of the Gaussian bleach profile over time is related to the dimensionality of the recovery process ( process .Data processing was performed in IgorPro (Wavemetrics) as follows:I values along its contour x.A previously described algorithm  was applI(x) data was fit to a 1D Gaussian by the least-squares method to extract the width, 2w, and amplitude as a function of time for each image.The D from the recovery process, the fitting parameter 2w is plotted vs. t, which yields a straight line of slope 2D.To estimate I(t) was plotted against t and fit to a single exponential using To estimate the mobile and immobile fraction, the intensity values of the bleached region was normalized according to mec-7(wy116) allele was isolated in genetic screen for mutants displaying defects in synaptic vesicle transport in the touch receptor neurons. We used TV1838 wyIs97  transgenic animals (wyIs97 drives expression of membrane-bound GFP (myrGFP) and the vesicle protein RAB-3 tagged by mCherry and reveals an accumulation of synaptic vesicles in a distal branch of the PLM neuron in the ventral nerve cord. Briefly, wyIs97 animals were mutagenized by exposure to ethane methanesulfonate (EMS) and screened for a loss of mCherry-tagged synaptic vesicles in the ventral cord synapses formed by the PLM neurons. The intensity of the synaptic patch was reduced nearly 50-fold in wy116 mutants compared to wild-type controls. The wy116 allele was mapped to the genomic interval containing mec-7 using standard genetic mapping techniques and sequenced to identify a missense, molecular defect replacing the threonine at position 409 with an isoleucine.The  animals  to visuauIs31[mec-17p::GFP] III; mutant, GN115 uIs31; mec-7(wy116); GN400 uIs31; unc-70(e524); GN514 uIs31; unc-70(s1502); GN119 uIs31ptl-1(ok621); GN643 uIs31ptl-1(ok621);unc-70(e524); GN644 uIs31; unc-70(e524); mec-7(wy116); EG4492 unc-70(s1502);oxIs95. Briefly, we used an AFS freeze substitution  apparatus to preserve worms in either 0.2% glutaraldehyde (GA) and LR-White or 1% OsO4 plus 1% Uranyl acetate in acetone with or without 3% water  using high-pressure freezing (HPF) followed by freeze substitution (FS), as previously described , 2007. T3% water  and embeL = 2 N a/T, where N, a, and T are the average number of microtubules per section, the length of the series, and the number of microtubule terminations in the series  models using Reconstruct  or TrakEe series . Microtu2 within a search window of 96 pixels2. Microtubule displacement was determined by searching for the maximal intensity correlation between the interrogation and search windows, with a threshold of 0.6.  The maWe used a discrete elastic rod model  to simulC  with the rod’s centerline. To model a twist deformation, an orthonormal material frame defined by the vectors 1d and 2d is assigned to each point on the centerline such that 1d and 2d span the plane normal to the centerline’s tangent, called the cross-section, while t lies tangent to the centerline. This material frame is adapted to the centerline and can rotate around the tangent and thus can describe a twisted configuration. The elastic energy per unit length (strain energy density H) of a deformed configuration is described by the components bending, twist and stretch energy:The Kirchhoff rod is represented by an adapted framed curve, which is tangent-aligned  is the dynamic space curve coordinate with s as the material coordinate and t = time. The tangent vector is defined as where ++ and based on the implementation of http://www.cs.columbia. edu/cg/elastic_coiling/. To evaluate how axon shape depended on axial stiffness, bending stiffness and shear modulus, we varied each modulus by a factor of ten. The range of values examined in the simulations were: (1) Bending stiffness: 2 < B < 2 and (2) Young’s modulus: 1 kPa < E < 10 kPa, which defines values for G and k according to We simulated friction with the extracellular environment as viscous drag using a Stoke’s drag on a cylinder in a low Reynolds number regime for every line segment of the discretized rod. The simulations were implemented in CFor normally distributed datasets, the sample size needed to detect a difference was calculated given the variance in the experimental data. Otherwise, sample sizes were not predetermined. Experimental parameters were varied randomly, and no sequence in data acquisition was applied. Behavioral experiments were conducted blind to genotype.F-test. We used Kolmogorov-Smirnoff or Jarque-Bera methods to test if our data were normally distributed and the modified Levene’s test of the median to test for equality of variances in non-normally distributed data.For TEM, sample size was limited by the extensive labor required to for sample preparation, imaging serial sections, aligning the image stacks, and analyzing the distribution of microtubules. Microtubule organization  was analyzed blind to genotype. Variance of microtubule number in different genotypes was tested using Fisher’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Caenorhabditis elegans Neurons" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Anna Akhmanova as the Senior and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal his identity: Michel Labouesse (Reviewer #1).Thank you for submitting your article "The Mechanics of Supercoiling in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This manuscript combines quantitative modeling and high resolution imaging techniques  to analyze how axons resist mechanical stresses. The authors reveal that axon shape is stabilized by combining the effects of three main actors: the actin-spectrin cytoskeleton protects against axial tension, microtubules against bending and the tau protein against torque. Their results bring significant novelty in understanding how mechanical constrains can affect axon shape and can be counteracted. Overall, the results are of extremely high quality. Conceptually, they bring a significant and important contribution to the understanding of how mechanical forces impact on the function of the nervous system.Essential revisions:This manuscript combines quantitative modeling and high resolution imaging techniques  to analyze how axons resist mechanical stresses. The authors reveal that axon shape is stabilized by combining the effects of three main actors: the actin-spectrin cytoskeleton protects against axial tension, microtubules against bending and the tau protein against torque. Their results bring significant novelty in understanding how mechanical constrains can affect axon shape and can be counteracted. Overall, the results are of extremely high quality. Conceptually, they bring a significant and important contribution to the understanding of how mechanical forces impact on the function of the nervous system.1) Title: The title doesn't really convey the key message. Please change the title for something more biologically appealing.2) The model. First, the Results start in a rather abrupt way with the presentation of the model without much context. It would be important for the lay neurobiologist to give some background, as very few readers will be familiar with the work of Alain Goriely. Ideally, the authors should start with some biological observation, and from there go on with their model. Second, it would be much easier to have the final section of the model assumptions and discussion in the main text (section 4 of the Supplementary notes). In this section, it would be important to give the range of the shear modulus values (as done for tension and stiffness). Please correct the many typos in that section.3) 4) unc-70(e524) in the presence of epidermal unc-70. In addition, do the axons still buckle when unc-70 is expressed in the epidermis? Pictures/analysis as in 5) unc-70(s1502) but not in the tetramer domain mutant unc-70(e524). Could the authors comment on that? An equally plausible explanation for that rescue could be that these animals are paralyzed, which as detailed in Hammarlund, Jorgensen and Bastianiet al., 2007) is sufficient to rescue some of the unc-70 axon phenotypes.Further, the authors show that epidermis expression of UNC-70 partially rescues the microtubule number and length in ALM neuron in the null mutation unc-70 mutation, even though these cells experience more mechanical deformations  mutant? Does expression of epidermal UNC-70 in the epidermis restore spectrin periodicity in axons?6) The periodicity of spectrin distribution is not very obvious in vivo. For instance, it is difficult to see α-spectrin periodicity in the ALM axon 7) 8) ptl-1 mutants that have epidermal expression of unc-70?9) Further, laser cutting experiment is not at all convincing, since the torque release in the 10) Data and Discussion on torque generation:unc-70 mutants or after Latrunculin A treatment. The parameter space derived from their model predicts that the formation of plectonemes through torque depends on tension and stiffness. To substantiate why axons do not form plectonemes, despite the appearance of torque in ptl-1(ok621), it would be important to determine the stiffness of ptl-1 axons and/or mec-7(wy116) mutants . The Results section on ptl-1 ends with a bold statement saying "organized into a twisted bundle, implying ptl-1 mutant axons have both decreased bending stiffness and increased torque". What is the basis for this statement? It would help to have in the text the constitutive equation accounting for it (if based on an equation).The authors present AFM data to show how stiffness is perturbed in The origin of torque generation should be discussed better. The authors suggest that MTs twist to maximize the length of contact lines. This suggests that it is related to the microtubules having 15 protofilaments and therefore having a supertwist. Does the pitch of twisting correspond with the pitch of the protofilament supertwist? Does this mean that 13 pf do not show this effect and that these finding cannot be translated to mammalian axons?11) Methods: It would be important to better explain in the main text how the shape analysis was quantified and done. The equation provided in the Methods is not very clear: (i) why sum from i=0 to i=N-n? (ii) my understanding of path length is that it measures a distance, and should thus be expressed in meters; if so, the <∆χ2> would overall be expressed in meters, correct? Essential revisions: […] 1) Title: The title doesn't really convey the key message. Please change the title for something more biologically appealing.We revised the title to encompass both the physical model for sensitizing axons to mechanical stress and the proteins we identify as key players. The new title is:C. elegans Axonsto Movement-induced Damage”“Genetic Defects in β-spectrin and tau Increase Torque-Tension Coupling and Sensitize 2) The model. First, the Results start in a rather abrupt way with the presentation of the model without much context. It would be important for the lay neurobiologist to give some background, as very few readers will be familiar with the work of Alain Goriely. Ideally, the authors should start with some biological observation, and from there go on with their model. Second, it would be much easier to have the final section of the model assumptions and discussion in the main text (section 4 of the Supplementary notes). In this section, it would be important to give the range of the shear modulus values (as done for tension and stiffness). Please correct the many typos in that section.eLife and pushed us to re-order the empirical and computational work. As the reviewers and editors can imagine, responding to this request has resulted in significant revisions to the entire text and all of the figures.As we understand it, this critique has two main components. First, the reviewers argue that the mechanical simulations original  are a diThe revised flow of ideas in the manuscript is as follows. The revised manuscript leads with the observed shape defects found in mutants carrying defects in actin-spectrin networks, the microtubule bundle, and both structures. We now provide both static and dynamic images of the most striking defects –9. The mSecond, the reviewers asked for model assumptions and parameter values evaluated in the simulations  to be incorporated into the text. This is a fine way to help readers place the model, its assumptions, and values for physical parameters into context. We added the relevant information to the text (Results) and refrain from publishing the full presentations of the simulation strategy, which is explained in greater detail in its original publication by Bergou et al., 2008, 2010. The new text:B, C, and k all depend on Young’s modulus, E, and the rod’s cross-sectional area, A, such that changes in one mechanical parameter also affect the others. […] Similar arguments can be made for shear modulus and tension, justifying the large parameter space explored in our simulations  ptl-1;unc-70 mutant axons and report the results in the text: “The defects in ptl-1;unc-70 mutants appeared at roughly regular intervals of 13 ± 0.5 µm  along the axon .We added a brief discussion of pearling instability to the Discussion:“Changes in tension have also been linked to pearling, a phenomenon in which tubes under tension form structures that resemble pearls on a string . Whereas pearling is driven by a competition between membrane tension and actin rigidity , the periodic defects we found in tau-spectrin double mutants occur due to an unbalanced torque-bend coupling, such that internal forces of the MTs are the driving force for the observed deformations.”4) him-4 mutants, illustrating the impact of the axon position on mechanical stress.  unc-70(e524) and unc-70(e524);him-4 double mutants, illustrating that buckling defects similar to those seen previously only in AVM  can occur in ALM neurons displaced towards the dorsal midline by loss of him-4 function. These results suggest that AVM and ALM are both sensitive to decreased axial tension and mechanical compression. Finally, we revised the schematic diagrams to more clearly illustrate axon position and note that the still images shown in We thank the reviewers for their attention to our presentation of axonal shape defects. We revised the figure to conform to this very helpful comment, including adding zooms. At the same time, it also made the previous composition of the figure unwieldy. To accommodate these changes, we reorganized the information into two separate figures. 5) unc-70(e524) mutants compared to wild-type and that expressing wild-type UNC-70 β–spectrin in the epidermis rescues  this effect  mutants and suggest testing this idea by STED imaging. To achieve this goal, we would need to visualize spectrin periodicity in vivo. Technical barriers prevent us from conducting this experiment. The tag we used to visualize β-spectrin would rescue unc-70(e524) in all tissues since it is a full-length fusion  expressed under the endogenous unc-70 promoter. Thus, we cannot perform STED imaging of β -spectrin in the mutant background either in vivo or in vitro. We used α-spectrin SPC-1::GFP to visualize spectrin periodicity in wild-type and unc-70(e524) dissociated neurons in culture  to  culture . Our effIn addition, do the axons still buckle when unc-70 is expressed in the epidermis? Pictures/analysis as in unc-70 mutants expressing the epi::UNC-70(+) transgene have reduced axon buckling compared to unc-70 mutants lacking this transgene. As a result, we chose not to reprise these findings in the present manuscript.Data addressing this comment were shown previously —Further, the authors show that epidermis expression of UNC-70 partially rescues the microtubule number and length in ALM neuron in the null mutation unc-70(s1502) but not in the tetramer domain mutant unc-70(e524). Could the authors comment on that? An equally plausible explanation for that rescue could be that these animals are paralyzed, which as detailed in Hammarlund, Jorgensen and Bastiani, 2007) is sufficient to rescue some of the unc-70 axon phenotypes.s1502 null allele and the e524 missense allele of unc-70. To reiterate, we observe that expression of the epi::UNC-70(+) transgene increases MT length and number in both unc-70 alleles, but that the effect size is bigger in the s1502 allele than in the e524 mutant . […] In light of these differences, we interpret the increased variance in MT as an indication that some long MTs persist in s1502 mutants because mechanical stresses are decreased in this genetic background and other MTs are shortened due to the loss of actin-spectrin networks.”“Related to the expected mechanical stresses generated in these animals, Furthermore, why are MTs in the AVM not shorter upon unc-70 mutation, even though these cells experience more mechanical deformations ? Does a unc-70 mutants. We have revised the text to reflect this interpretation , included a quantification of the buckling wavelength  establish that MTs in wild-type AVM neurons are much shorter than those found in ALM and that the short MTs are less sensitive to the compression-induced buckling present in velength , and remStrikingly, the average MT length in the AVM is also more than twice shorter than previously published. What is the explanation for this?Panel D of The data from 6) The periodicity of spectrin distribution is not very obvious in vivo. For instance, it is difficult to see α-spectrin periodicity in the ALM axon We agree that spectrin periodicity is difficult to detect in vivo. This is because the ALM is engulfed by the epidermal cell and that both cells express SPC-1::GFP, which we used to visualize spectrin periodicity. We suspect that the two cells are too close to adequately image independent of one another, even with super-resolution methods. This complication was evident in the PSD Original  that is Could the authors explain why there are two peaks in The referees raise an interesting point. The PSD in the original Besides, how do the authors explain that the localization of α spectrin is not perturbed in the unc-70(e524) mutant?unc-70(e524) missense and unc-70(s1502) null mutants. In particular, we show that e524 mutants retain a periodic distribution of α–spectrin but that the s1502 mutants do not. Because the s1502 allele is null and is expected to lack b-spectrin, these observations imply that no periodic actin-spectrin network is formed in this mutant even α–spectrin protein is retained. Consistent with this idea, STED images in s1502 mutants are very similar to those observed in cells treated with the actin depolymerization agent, Latrunculin A  is a point mutation mapping to the linker between the 16th spectrin repeat and the tetramerization domain. It is thus likely, that this defect does only partially interfere with spectrin assembly, but makes the network mechanically less rigid. Interestingly, we also observe a reduction in length of the periodic structure, indicative of a subtle, but consistent effect suggesting a change in mechanics.Further, Does expression of epidermal UNC-70 in the epidermis restore spectrin periodicity in axons?oxIs95 transgene to drive expression of wild-type UNC-70 in the epidermis in vivo. As we cannot resolve the periodicity of α-spectrin in intact animals reliably due to background fluorescence from neighboring tissues, we removed all STED images taken from fixed animal from the revised manuscript.This comment overlaps with point 3 above and could only be addressed by analyzing spectrin periodicity in the presence and absence of the 7) We agree with the referees that a quantitative description would be a terrifically useful addition to the study. Accordingly, we have now quantified the flow field between selected cross-sections showing a clear rotation around the central bundle axis Video 17.Could the authors explain in more detail how the angle between MTs was determined (a scheme would help), and how the twisting of the axon and MTs would arise?mec-7(wy116) β-tubulin and ptl-1(ok621) tau mutants”, fourth paragraph).We added a schematic to Does the observed twist depend on the animal moving, and would it be observed in a fully paralyzed mutant?If the reviewers are referring to the twisted MT phenotype, it is detected in fixed samples . We also8) We reorganized the paragraph as suggested by the reviewers as part of the global reorganization.9) Further, laser cutting experiment is not at all convincing, since the torque release in the ptl-1 mutants. We note that ptl-1 mutants express wild-type unc-70 in neurons and epidermis—were the reviewers thinking instead of how epidermal expression of UNC-70(+) affects the plectonemes observed in ptl-1(ok621);unc-70(e524) double mutants? This is an interesting question, especially since we have previously shown that expressing UNC-70(+) partially suppresses movement-induced buckling in unc-70(e524) mutants .  triple mutants and imaged the resulting animals under the same conditions used to observe ptl-1;unc-70 double mutants. We found that the epidermal re-expression of unc-70 did not abolish the development of the plectonemes, although we observed a subtle effect on plectoneme size. We included these data in This comment appears to encompass two subpoints. Firstis the question of whether or not epidermal expression of UNC-70(+) would affect ptl-1;unc-70 double mutant axons and asked us to provide clearly examples and to improve the presentation of this result. We have now repeated these experiments and added new example videos to this manuscript, clear showing three dimensional re-arrangements and rotations  Data and Discussion on torque generation:The authors present AFM data to show how stiffness is perturbed in unc-70 mutants or after Latrunculin A treatment. The parameter space derived from their model predicts that the formation of plectonemes through torque depends on tension and stiffness. To substantiate why axons do not form plectonemes, despite the appearance of torque in ptl-1(ok621), it would be important to determine the stiffness of ptl-1 axons and/or mec-7(wy116) mutants .ptl-1 and mec-7 mutants and included these data in unc-70(e524);ptl-1(ok621) double mutation, has a lower E as well as a low tension  single mutation on tension is preserved in the double.We have now repeated the AFM experiments on the The Results section on ptl-1 ends with a bold statement saying "organized into a twisted bundle, implying ptl-1 mutant axons have both decreased bending stiffness and increased torque". What is the basis for this statement? It would help to have in the text the constitutive equation accounting for it (if based on an equation).We have now included a paragraph relating the number and cross-section of the MTs within the bundle to its mechanics. We write to inform the reader that the anatomy and geometry of the bundle relates to its mechanics according to the references cited therein:“Given that bundle stiffness is expected to be proportional to the number of filaments in the bundle , this finding suggests that bending stiffness is decreased in both of these mutants. […] A decrease in area and in the number of cross-links is expected to decrease the bending stiffness of the bundle .”The origin of torque generation should be discussed better. The authors suggest that MTs twist to maximize the length of contact lines. This suggests that it is related to the microtubules having 15 protofilaments and therefore having a supertwist. Does the pitch of twisting correspond with the pitch of the protofilament supertwist? Does this mean that 13 pf do not show this effect and that these finding cannot be translated to mammalian axons?We have now included a discussion about 13-protofilament microtubules in the main text. In essence, we believe that our results translate to all MT, as every MT possess a helical surface lattice, independent of its protofilament number. We also added a citation, showing the development of similar coils in cultured, mammalian axons . We added the following sentence to the Discussion:C. elegans TRNs, but also to the more common 13-protofilament MTs found in mammalian neurons. Consistent with this idea, MTs bend and deform into similar structures under compressive forces in cultured hippocampal neurons  and form twisted, hexagonal bundles in certain in vitro preparations .”“As all microtubules possess a helical surface lattice , this mechanism is independent of the number of protofilaments per microtubule and applies not only of the unusual 15-protofilament MTs present in 11) Methods: It would be important to better explain in the main text how the shape analysis was quantified and done. The equation provided in the Methods is not very clear: (i) why sum from i=0 to i=N-n? (ii) my understanding of path length is that it measures a distance, and should thus be expressed in meters; if so, the <∆χ2> would overall be expressed in meters, correct?We have now included a detailed description of the shape quantification in the main text:“To enable quantitative comparisons, we devised a method to quantify the neurite’s deviation from the straightest possible path[…] In this computation, a perfectly straight neuron has an effective constant D=0 and all deviations from straightness increase D, regardless of the nature of the morphological defect (see Methods).”The equation and method used to quantify the randomness is conceptually very similar to the formalism to quantify the diffusion constant by calculating its mean squared displacement average over increasing time windows. In our calculation, instead of averaging over increasing time windows, we average over increasing spatial windows. The advantage of our formalism is that it is very sensitive to local deviations from a straight line (e.g. due to buckling and shape defect), but less sensitive to non-local deviations, e.g. neuron curvature due to changes in body posture.2/length, which means it is the scale of a length.We have also included a detailed description of the underlying mean squared deviation (MSD) formalism in the Methods . The units for the spatial MSD are length