In the last few decades, Drosophila melanogaster emerged as a versatile model for studying neurodegenerative diseases, including ALS. In this review, we describe the different Drosophila ALS models that have been successfully used to decipher the cellular and molecular pathways associated with SOD1, C9orf72, FUS, and TDP-43. The study of the known fruit fly orthologs of these ALS-related genes yielded significant insights into cellular mechanisms and physiological functions. Moreover, genetic screening in tissue-specific gain-of-function mutants that mimic ALS-associated phenotypes identified disease-modifying genes. Here, we propose a comprehensive review on the Drosophila research focused on four ALS-linked genes that has revealed novel pathogenic mechanisms and identified potential therapeutic targets for future therapy.Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease characterized by the progressive degeneration of upper and lower motoneurons. Most ALS cases are sporadic but approximately 10% of ALS cases are due to inherited mutations in identified genes. ALS-causing mutations were identified in over 30 genes with  A(TARDBP) .C9orf72 gene was identified as a new cause of ALS and frontotemporal dementia (FTD) [C9orf72 gene represents the most common genetic cause of ALS [FUS and TARDBP encode two DNA/RNA-binding proteins, which play distinct roles in transcription, as well as numerous roles in RNA metabolism, including splicing, stability, and transport [TARDBP encodes the TDP-43 protein, which is mainly nuclear and shuttles between the nucleus and the cytoplasm. Nuclear depletion and cytoplasmic aggregation of TDP-43 are found in most if not all ALS patients independently of the mutated status of TDP-43, making it a hallmark of the disease [The antioxidant enzyme SOD1 was the first gene linked to fALS in 1993 . In 2011ia (FTD) ,22. Curre of ALS . C9orf72e of ALS , whose pe of ALS . Lastly,ransport . FUS is ransport , belongiransport . In 2009ransport ,30. TARD disease . However disease .Drosophila melanogaster is a model easy to handle, cost-effective, with a short lifespan and a fully sequenced genome since 2000 [Drosophila is a powerful genetic model with several genetic tools, such as the upstream activating sequence (UAS)/Gal4 system  or gene-switch systems [Drosophila ortholog [Drosophila has emerged as a powerful genetic model for studying several neurodegenerative diseases   phenotypes were unsurprising for a ubiquitous housekeeping enzyme involved in detoxification and remained in the line with predictions and aging model. More unexpected were the consequences of dSod1 gain of function (GOF). Transgenic Drosophila lines carrying an additional copy of dSod1 were constructed using random P element insertion [In  in 1989 . Many stobserved ,50,51. Tnsertion . These fnsertion .hSOD1 and the fALS [SOD1 gene was considered. Indeed, to date, about 200 ALS-associated mutations in hSOD1 have been described [G93A  showed an identical enzymatic activity compared to wildtype protein [mSOD1 knockout mice [hSOD1 induce the misfolding of the protein and confer new noxious properties. This toxic GOF is worsened by the ability of misfolded hSOD1 to spread from cell to cell causing the propagation of the disease [In 1993, the discovery of the genetic linkage between escribed ; most of protein . In mice protein ,61. In aout mice  strength disease ,71,72,73dSod1-null mutants showed a severe reduction in lifespan. In a dSod1-null background (x16/sodx39dSod1), expression of WThSOD1 under the control of endogenous cis-regulatory dSod1 sequences fully rescued the lifespan reduction as dSod1 expression did. In this genetic background, the expression of different fALS-related hSOD1 mutant alleles  showed a partial rescue of the lifespan compared to x16/dSod1sodx39 flies. However, the lifespan of fALS-related hSOD1 flies was shortened compared to flies expressing WThSOD1 allele. This lifespan reduction was coupled with an early drop of negative geotaxis performance in line with pathological phenotypes [Drosophila Sod1. Drosophila models were, therefore, generated using the UAS/Gal4 system [hSOD1 transgenes  showed that misfolded hSOD1 protein was produced in muscles. This expression led to shortened lifespan, drop in motor activity, and mitochondrial impairment [G93AhSOD1 transgene expression in the nervous system. Interestingly, when hSOD1 mutant forms were cell-autonomously expressed by motoneurons, glial cells exhibited a stress response as evidenced by the expression of the heat-shock protein 70 (HSP70) [N-methylamino-l-alanine) resistance study. BMAA is a neurotoxin found in cycad seeds that causes Guam disease, an ALS–Parkinsonism dementia complex [Drosophila motoneurons or glial cells led to different results. Expressed selectively in glial cells, the sensibility to BMAA was increased with a loss of motor performance over time. When hSOD1 constructs were expressed in motoneurons or in both motoneurons and glial cells, the resistance to the neurotoxin was enhanced, as well as the motor functions [These flies displayed progressive motor function deterioration over time. Contrary to what is observed in vertebrates, no neuronal cell death has been demonstrated in the adult ventral nerve cord where the motoneuron cell bodies are located. Therefore, ALS  disease ,79,80, ws of ALS ,70,81,82s of ALS ,84. Indepairment  (Table 2 (HSP70) . The rol (HSP70) ,86,87,88 (HSP70) ). Consis (HSP70) ,91,92. T complex . The expunctions . The mecunctions ,95,96.dSod1 and introduce ALS-related mutations at conserved residues in dSod1, thereby creating dSod1G85R, dSod1H71Y, and dSod1H48R mutants [G85RdSod1 homozygous died at the pharate stage and did not emerge properly. Surprisingly, despite the severe locomotor defects observed, no motoneuron death was detected in larvae or adult flies. Interestingly, all mutants produced significant amounts of dSod1 protein, and expression of wildtype dSod1 partially saved the G85R/G85RdSod1 phenotypes, leading to the hypothesis that both toxic GOF and LOF are combined to explain the observed ALS-associated phenotypes. Further study of G85RdSod1 mutants at the neuromuscular junction (NMJ) level showed that both the number of boutons and the neurotransmission were reduced in the mutant, at the pharate stage [G85RdSod1 displayed a clear crawling impairment that was not associated with a disturbing phenotype at the NMJ level. Electrophysiological recordings of NMJs and motoneurons did not show any difference in post-synaptic excitatory events between G85RdSod1 and wildtype flies, although motoneurons exhibited a slightly reduced excitability. The crawling behavior is generated by the central pattern generator network, an innate neural circuit composed of interneurons and motoneurons that generate rhythmic motor output. Sensory inputs play a role in adapting locomotor activity to external cues. Extracellular recordings and deafferentiation experiments showed that defective sensory feedback lead to reduced locomotor activity in G85RdSod1 larvae [The overall literature on the study of hSOD1 ALS-associated forms has shown that the phenotypes observed were not only dependent of the transgene expression level but were also related to the cellular type targeted. To overcome these difficulties and increase the understanding of the mechanisms leading to the toxicity of the SOD protein in ALS, recent studies chose to express hSOD1 at an endogenous level. For this, they used the ends-out homologous recombination strategy to replace wildtype  mutants . In homote stage . EarlierR larvae   produced from plants and known for its various properties including antioxidant potential [Drosophila expressing hSOD1G85R in motoneurons. LA was shown to moderate the neurotoxicity, extending lifespan and improving motor activity, in hSOD1G85R flies [Drosophila ALS context, Orz increased HSP70 expression and alleviated oxidative damage [otential  has been5R flies . In the 5R flies . In the e damage . These rDrosophila could help to understand the mode of propagation of misfolded hSOD1, as well as the relationships between different cell types .Until now, studies concerning hSOD1 have focused on major phenotypes , but a more detailed analysis at the cellular level of the hSOD1 actions could provide answers on the pathways leading to the pathology. Moreover, future studies using 4C2) HRE in the C9orf72 gene was identified as a new cause of ALS [C9orf72 gene produces three alternative spliced transcripts [4C2) repeat is present in the promoter region (for transcript variant 2) or within the first intron (for transcript variants 1 and 3) (C9orf72 HRE have been classified into three nonexclusive categories: (1) loss of C9orf72 function through haploinsufficiency, (2) sequestration of proteins by HRE-induced RNA foci, and (3) toxic GOF induced by dipeptide repeat proteins.In 2011, intronic GGGGCC  . To dateC9orf72 gene decreases the levels of messenger RNA (mRNA) [C9orf72 LOF in Zebrafish [Caenorhabditis.elegans [C9orf72 gene [C9orf72 function may exacerbate the repeat-dependent gain of toxicity [C9orf72 function may be involved in the disease. The Drosophila genome does not contain a C9orf72 ortholog gene [C9orf72 LOF in this organism.Several studies have shown that the HRE in A (mRNA) ,101 and A (mRNA) ,103 in pebrafish  and Caen.elegans  induced f72 gene . Howevertoxicity , suggestlog gene , making C9orf72-linked ALS patients is the presence of RNA foci [Drosophila, it was found that components of the Drosophila DRB sensitivity-inducing factor (dDSIF) and Drosophila polymerase-associated factor 1 (dPAF1) complexes, two regulators of RNA polymerase II, are selectively required for the transcription of the expanded (G4C2) repeats [4C2)10 repeat RNA with mouse spinal cord lysates and identified Purα as a protein able to specifically bind to the repeat [4C2)30 repeat cloned upstream of the translation start of GFP to express it in the developing eye. This induced a rough eye phenotype, revealing the toxicity of the expanded repeat that was not observed when the same construct carrying only 3 repeats was used. Overexpression of Purα with the (G4C2)30-GFP construct suppressed the neurodegeneration induced by the (G4C2)30-GFP transgene, supporting the idea that Purα function was attenuated in the presence of the expanded (G4C2) repeat. When the (G4C2)30-GFP construct was expressed in motoneurons using the OK371-Gal4 driver, around 50% adult eclosion failure and locomotion defect in 28 day old flies were observed. In addition, larval NMJs showed a reduction in active zone number. These phenotypes were suppressed by the overexpression of Zfp106, a zinc finger protein that binds specifically to a (G4C2)8 repeat construct [A hallmark found in different tissues, including motoneurons, of RNA foci . These R repeats ,111. Inde repeat . Purα ise repeat . The autonstruct . Interes4C2 HRE RNA forms hairpin and G-quadruplex structures that have the potential to recruit and sequester proteins. One such example is RanGAP (Ras-related nuclear GTPase-activating protein) that binds preferentially the sense RNA quadruplex of the HRE [4C2)30-GFP expression in the developing eye. This was also the case when eye-expressing (G4C2)30-GFP flies were fed with 5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4), a porphyrin compound that destabilizes RNA G-quadruplex tertiary structures. This indicates that the G-quadruplex structure was required for the recruitment of RanGAP on (G4C2) HRE RNA [It was shown that G the HRE . RanGAP  HRE RNA .4C2) expanded repeat RNAs, which generally aggregate as nuclear RNA foci, also localize to neuritic granules that were actively transported along neuronal processes [Drosophila have a complex dendritic branching pattern that was altered by the expression of a (G4C2)48 RNA repeat. The overexpression of dFMR1 or Orb2, two transport granule components that regulate local translation, enhanced the dendrite branching defects, while their RNAi-induced knockdown rescued the phenotype [4C2) HRE RNAs in neuronal processes.Burguette et al. found that (Grocesses . The prerocesses . The clahenotype . This suC9orf72 minigene containing (G4C2)160 repeat flanked by human intronic and exonic sequences [Despite the putative sequestration of RBPs by HRE RNAs, several studies have demonstrated that RNA foci per se were not toxic. Tran et al. used a equences . When ubC9orf72 splice variant, the (G4C2) repeat is present in the promoter region or within the first intron  expansion undergoes repeat-associated non-AUG (RAN) translation from both sense and antisense transcripts to produce five dipeptide proteins (DPRs) [4C2) expansion with alternative codons [36 and (GR)36 were toxic but not (GA)36 or (PA)36, indicating that only arginine-rich DPRs were toxic. When 100-copy DPRs were expressed, the toxic effect of arginine-rich DPRs was exacerbated but the other DPRs did not show toxicity. Despite its intronic localization, the (Gs (DPRs) ,119,120.e codons . Upon ey50 in glutamatergic neurons, including motoneurons, developed normally but were unable to escape out of the pupal case due to the absence of movement [50 or poly(PA)50 developed normally [50 induced eye degeneration that was not observed with ATG-driven GFP-tagged poly(GA)50 or poly(GP)47 constructs [100 but not poly(GA)100 induced a decrease of presynaptic area, as well as a reduction in active zone number [The toxicity of arginine-rich DPRs was also demonstrated by several other studies. For example, Drosophila expressing poly(PR)movement . In contnormally . The expnstructs ,124. At e number .80 had a diffuse cytoplasmic localization in Drosophila salivary gland cells, while (GA)80 formed cytoplasmic inclusions [80 and (GA)80 were co-expressed, part of (GR)80 formed cytoplasmic inclusions, suggesting that (GA)80 recruited (GR)80 into these inclusions. Of note, the same observation was found in induced pluripotent stem cell (iPSC)-derived human neurons [80 induced cell loss at the wing margin that was partially suppressed by the co-expression of (GA)80. However, this protective effect of (GA)80 was not observed in the eye [Several DPRs are found in ALS patients; however, it remains to be determined whether some DPRs can interact with others to modulate toxicity. Indeed, one study has shown that (GR)clusions . Interes neurons . The exp the eye . Thus, i100 but not (GR)100 or (PR)100 was detected in fly brains after 3 days of expression in ORNs [100 was detected in axons and synaptic terminals of ORNs. Interestingly, the spreading was increased when a longer construct was used. It would be interesting to combine several DPRs to determine whether poly-GA may recruit other DPRs allowing them to spread between neurons.The transcellular spreading of misfolded proteins is thought to participate in the clinical progression of several neurodegenerative diseases. To answer whether DPRs have the ability to spread, mCherry-tagged DPRs were expressed in olfactory receptor neurons (ORNs) that target their axonal projections in fly brain. When 36 copies of (GA), (GR), or (PR) were used, no spreading was observed. In contrast, (GA) in ORNs . Accordi50 and GFP-(GR)50 and included some ALS-related RBPs, such as TDP-43 and FUS, for example, suggesting that arginine-rich DPRs may alter RNA metabolism. Moreover, these common interactors showed enrichment in proteins containing low-complexity sequence domains (LCDs), which mediate the assembly of membrane-less organelles. By using an RNAi genetic screen targeting the orthologous genes in Drosophila, the authors found that most of the genetic modifiers of GFP-(PR)50-induced toxicity were components of membraneless organelles such as nucleoli, the nuclear pore complex, and stress granules [58-induced eye degeneration identified 18 modifiers that play a role in nucleocytoplasmic transport, suggesting that nuclear retention of mRNA may be a cause of toxicity [25, Boeynaems et al. showed that nucleocytoplasmic transport was implicated in the pathogenic mechanism of C9orf72 HRE [4C2)36 repeat or (GR)100 pan-neuronal expression. As eIF1A plays an important role in translation initiation, these data suggested that translation machinery might be affected by arginine-rich DPRs.In a way to understand how arginine-rich DPRs may cause toxicity, Lee et al. analyzed the interactome of GFP-tagged DPRs . Among tgranules . This sttoxicity . By perfrf72 HRE . Anotherrf72 HRE . This st80 expression in Drosophila muscles induced alteration of indirect flight muscles leading to defects in wing posture [80 within mitochondria where they interacted with components of the mitochondrial contact site and cristae organizing system, leading to mitochondrial defects. Thus, it appears that poly(GR) may cause toxicity by interfering with mitochondrial functions, at least in muscle cells.A recent study showed that poly(GR) posture . These aC9orf72-associated toxicity that may highlight novel therapeutic targets. For example, reducing the function of components of the specific machinery required for transcription of expanded repeat RNA, such as dDSIF and dPAF1 complexes, should reduce levels of toxic expanded RNA, as well as the production of toxic DPRs [Drosophila expressing (G4C2)36 repeats [+/H+ antiporter that rebalances mitochondrial matrix ion levels, opening the way to future potential therapeutic strategies [Altogether these studies have identified several pathways of xic DPRs ,111. Anoxic DPRs . Furtherxic DPRs . When ad repeats , confirmrategies .Fused in sarcoma, also known as translocated in liposarcoma (TLS), is a DNA/RNA-binding protein ubiquitously expressed. ALS-causative mutations in this RBP were discovered in 2009 ,30. In tAs an RBP, FUS has a pivotal role in many aspects of RNA metabolism and processing, including RNA splicing ,137, traThe mechanisms underlying the cause of neurodegeneration in FUS-ALS patients are still unknown even if numerous hypotheses have been made. Hence, mislocalization of FUS mutant forms in the cytoplasm leads to a disruption of FUS nuclear functions, such as transcription regulation or mRNA processing, and produces a LOF phenotype ,150,151.Drosophila, the first studies showed that eye-overexpression of wildtype FUS or different ALS-related mutated forms led to progressive neurodegeneration of the photoreceptors [In eceptors ,158,159.As ALS is characterized by the degeneration of motoneurons, the action of FUS expression in this specific neural population was considered. When expressed in motoneurons, all forms of FUS (wildtype and ALS-related) led to a deficit in locomotion at the larval stage followed by a lethality occurring at late pupal stage ,158 and . At the Adult eclosion defect and late pupal lethality were also observed using a general neuronal driver as Elav-Gal4. Nevertheless, use of inducible Elav-Gal4-GS line has allowed showing that FUS mutant forms induced a drastic climbing decline associated with a reduced lifespan, more severe than wildtype FUS did . Thus, tThe cellular localization of FUS has also been studied. As in ALS patients, mutated forms of FUS were both found in the nucleus and mislocalized in the cytoplasm, whereas wildtype FUS was always detected in the nucleus ,30. ALS-cabeza (caz) gene. caz is the only ortholog of FUS in Drosophila [1caz mutants appeared morphologically normal but displayed an adult eclosion defect, and the resulting adult escapers showed reduced lifespan and deficit in locomotion  acted on the survival to adulthood of 1 Drosophilacaz with no effect on the lifespan and locomotion of adults [To decipher FUS functions, studies have focused on the role of the osophila . The cazomotion  localized outside of the nucleus but did not exhibit any phenotype when expressed in the eye or in motoneurons. Moreover, addition of an NLS sequence in the FUSΔ32 construct led to a nuclear localization of the protein associated with locomotor defect and eye degeneration phenotypes [Both Caz and FUS are found in the nucleus in the nervous system. To address the role of FUS localization in toxicity, FUS constructs deleted for the NES domain were engineered in both wildtype FUS and ALS-related mutants. While FUSytoplasm . The douto arise . Howeverenotypes . These ccabeza points out that FUS could autoregulate its own expression in Drosophila [Interestingly, the observation that the overexpression of wildtype FUS or mutant forms in the motoneurons led to a drastic downregulation of osophila ,168. Morosophila , meaningcaz and FUS and (ii) the idea that the expression level of FUS is critical to trigger the neurodegeneration process [These results strengthen (i) the notion of conservation between  process ,164,168.WT or ALS mutant FUSP525L led to mitochondrial damages in transgenic flies. Compared to control condition, mitochondria were smaller, and the number of larger ones decreased. With FUS mutant overexpression conditions, the phenotype was more pronounced [P525L or CazP398L mutants localized in the cytoplasm and were found at the synaptic projections of the da neurons, which were altered. All forms of FUS and Caz (wildtype or mutant) impaired axonal transport of the synaptic vesicles. A decrease in the number of synaptic mitochondria in da neurons was also observed. The expression of all forms of FUS and Caz induced an increase in the frequency of calcium transients in da neurons [Mitochondria continually undergo fission and fusion processes; the breakdown of this balance is decisive in neurodegenerative diseases. Mitochondria disruption has been extensively reported from ALS patient studies ,183 , a marker of lysosomes that was upregulated [The nucleus is compartmentalized in membraneless intranuclear compartments collectively named nuclear bodies (NBs). NBs include Cajal bodies, nucleoli, nuclear speckles, and paraspeckles that are dynamic structures responding to stress and controlling gene expression. NBs are the location of RNA biogenesis and maturation, and they are involved in the assembling of ribonucleoprotein complexes or in the retention of proteins ,190,191.t hnRNPs ,198,199.the NMJs . In addipression . The samytoplasm . In thisegulated ,200. ThuAs mentioned above, FUS is composed of different domains whose functions have been revisited in recent years. Previously, the C-terminal domain was the focus of most studies because most ALS mutations cluster in the NLS sequence and were known to disrupt the nuclear localization of FUS. Revisiting the FUS amino-acid sequence showed that the N-terminal part contains a prion-like domain followed by a glycine-rich and an arginine–glycine–glycine repeat sequence (RGG) . These tIn motoneurons, expression of wildtype FUS led to pupal death. Flies cannot emerge from the pupal case and the few escapers observed display a soft cuticle and unexpanded wings that are characteristics of an immature phenotype . These pAs already reported, mutations in the PY-NLS domain lead to mislocalization of FUS in the cytoplasm and induce a strong eye degeneration phenotype compared to wildtype FUS . In motoAn in vitro study showed that the LCD of FUS is involved in the self-assembly process . In motoThe lack of phenotype observed with the C-terminally truncated FUS mutant could be explained by some differences in the LCD of Caz and FUS proteins. Indeed, the QGSY domain is absent in Caz protein . Thus, Ncis to mediate FUS toxicity [FUS is a protein prone to aggregate and, in patients, FUS is found in cytosolic aggregates. In fact, the LCD of FUS shares similarity with the yeast prion protein ,208. Howtoxicity . In vitrtoxicity ,209 and toxicity ,211,212.toxicity . In the toxicity ,215,216)Drosophila, motoneuron expression of a FUS protein containing a RGG2 domain mutated in all arginines rescued the FUS-induced toxicity like the deletion of the entire RGG2 domain. Hence, RGG2 and LCD regions play a crucial role in FUS aggregation.In ALS patients, FUS co-localizes with SG markers , where iDrosophila, and hypophosphorylated forms of the protein alter FUS solubility properties, causing toxicity. Indeed, insoluble FUS corresponds to hypophosphorylated forms of the protein that mediate toxicity independently of inclusion formation [Drosophila arginine methyltransferase proteins (DARTs) [PTMs, which are known to regulate protein structure and function , influenormation . Moreoveormation . This suormation ,226,227.ormation ,206. The (DARTs) . More pr (DARTs) ,229,230. (DARTs) . Convers (DARTs) . AccordiDrosophila has always been a system model used to search for toxicity-modifying genes. Several genetic screens have been done to understand the mechanisms leading to motoneuron neurodegeneration using either Caz or FUS genetic background. This approach has enabled finding different interacting proteins involved in distinct cellular processes, all implied in FUS-induced neurodegeneration.caz-knockdown induced phenotypes (rough eye and locomotor defect) has allowed the identification of ter94  [ter94 function enhanced the caz-knockdown phenotypes, and conversely the expression of ter94 in eye or neurons can rescue the neurodegenerative phenotype observed in caz-knockdown flies [In an attempt to understand the mechanisms that lead to FUS toxicity and neurodegeneration, the use of in, VCP) . Ter94 iin, VCP) ,233,234.wn flies . Intereswn flies . Recentlwn flies ,237. Indcaz with Xrp1, a DNA-binding protein involved in gene expression regulation, chromatin remodeling, and DNA repair [caz mutant phenotypes [caz mutant background, and this genetic interaction is dependent on the Xrp1 DNA-binding domain. These observations led to the hypothesis that, in caz mutants, increased Xrp1 expression could induce gene expression dysregulation causing neurodegeneration [Xrp1 ortholog has yet been found in mammals. Nevertheless, in the context of mutant FUS expression, Xrp1 downregulation rescues the ALS-induced phenotype, somehow suggesting conservation in the gene expression dysregulation mechanism leading to ALS pathology.More recently, the same strategy enabled highlighting the link of A repair ,239. In enotypes . Xrp1 exneration . No Xrp1caz genetically interacts with different genes involved in the piRNA biogenesis such as Piwi, Aubergine, and Argonaute3 [Drosophila brain, and Aubergine (Aub) is involved in piRNA biogenesis in neurons [caz knockdown many abnormal pre-piRNAs were found. Aub overexpression enhanced the neuronal defects induced in this genetic background and increased the cytoplasmic localization of Caz [gonaute3 . piRNAs gonaute3 ,242,243. neurons ,245. In n of Caz . Indeed,Pink and Parkin, two genes already described to modify FUS toxicity. These genes act in a common pathway to target damaged mitochondria toward an autophagic pathway to control mitochondrial quality [Pupal lethality and reduced adult eclosion rate phenotypes induced by FUS expression in neurons secreting the bursicon neuropeptide  are particularly suitable for the design of genetic screens . This ap quality . Downreg quality ,186. Can quality . cabeza knockdown-induced phenotypes [The rough eye phenotype induced by FUS expression was also used as a readout for genetic screens. Recently, this paradigm led to the identification of the Hippo pathway as a modifier of FUS-induced neurodegeneration . Hippo senotypes , strengtDrosophila has allowed gaining insight into the understanding of the mechanisms implied in FUS-induced neurodegeneration. However, many data seem contradictory and deserve to be investigated de novo in light of new data. For example, very recently, FUS was found to be a bicistronic gene coding for two proteins [Drosophila [proteins . The FUSosophila , a more TARDBP gene, was identified as the major component of the ubiquitin-positive cytoplasmic inclusions found in ALS patients [TARDBP were linked to sporadic and familial ALS [TARDBP mutations have been identified [In 2006, TDP-43, which is encoded by the patients ,253. In lial ALS ,258,259.entified , which aentified . Importaentified . Drosophila an ideal organism to decipher the function of TDP-43. The Drosophila ortholog of TARDBP is TBPH, and both LOF and GOF approaches have been modeled in Drosophila to gain insights into TDP-43 toxicity.TDP-43 is a member of the hnRNP family that is ubiquitously expressed. TDP-43, which acts as a transcriptional repressor, has been implicated in a wide variety of RNA metabolic processes including splicing, stability, and transport . TDP-43 TBPH, which is expressed throughout development [TBPH is mainly nuclear but was also found in the cytoplasm [TBPH in vivo, several groups have created TBPH-null mutant flies  [TBPH mutants [TBPH mutant larval NMJs [TBPH [TBPH is required to maintain glial integrity and glutamate receptor distribution. Interestingly, pan-glial expression of the glutamate transporter dEAAT1 in TBPH mutant restored the normal distribution of glutamate receptors but not the glial wrapping defects, suggesting that TBPH acts on different pathways in glial cells [The presence of TDP-43 aggregates in glial cells of ALS patients ,284,285 em (CNS) ,271. HowTBPH mutants display an upregulation of retrotransposable elements in neural tissues [TBPH mutants have a defective retrotransposon silencing. This was likely due to the reduced level of Dicer-2 observed in TBPH mutant as pan-neuronal expression of Dicer-2 prevented the upregulation of retrotransposable elements [It was also described that  tissues . By usinelements .TBPH mutants was the decrease in dendritic branching of larval sensory neurons [TBPH RNAi specifically in sensory neurons  analysis [Another phenotype described in  neurons . The sam neurons , indicatanalysis .TBPH mutant adult escapers also have ectopic bristles and sensory neurons on their notum [TBPH. The precise generation of SOP cell number is regulated by microRNA (miR)-9a [TBPH mutant [TBPH modulates miR-9a production to ensure proper neuronal specification. Another report has described that the wing phenotype induced by miR-1 overexpression was enhanced in heterozygous TBPH flies, suggesting that TBPH may dampen miR-1 activity [ir notum , indicat(miR)-9a , which hH mutant . Thus, iactivity .TBPH in different tissues . Furthermore, one report showed that TBPH and TDP-43 share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983 and Squid/Hrp40/CG16901 [TBPH/TDP-43 with some hnRNPs is conserved during evolution. Moreover, TDP-43 and TBPH were shown to be interchangeable regarding their nucleic acid binding specificity and their role in splicing [Several studies have shown that  defects ,267,296. defects . This cl/CG16901 . These t/CG16901 . Thus, fsplicing .TBPH and TDP-43 GOF experiments have demonstrated that a high level of TBPH or TDP-43 is very toxic  induces dramatic defects . When TDP-43Nter-RAVER1 was expressed in motoneurons of TBPH mutants, it highly extended lifespan [Nter-RAVER1 construct carrying two mutations in the RRM1 that abolish RNA binding had no effect [Nter-RAVER1 was due to TDP-43 binding onto its target genes. Altogether, these data strongly suggest that a major function of TBPH is splicing repression, which is very likely involved in the drastic phenotypes induced by loss of TBPH.RNA-sequencing of pression . To go flifespan . On the o effect . This coWT or TDP-43A315T induced mitochondrial damages including reduced size and abnormal cristae [WT or TDP-43A315T [WT or TDP-43A315T GOF induced an activation of the mitochondrial unfolded protein response (mitoUPR), characterized by the increased expression of known genes involved in mitoUPR, including Lon, the Drosophila ortholog of LonP1 [Lon silencing exacerbated TDP-43-induced retinal degeneration and mitochondrial damages, as well as increased the level of mitochondrial TDP-43 without affecting the cytoplasmic level [Abnormal fragmentation of mitochondria was described in the adult brain upon pan-neuronal expression of TDP-43 . Highly  cristae . In addi-43A315T . Pan-neuof LonP1 . LonP1 iic level . This sugypsy RTE, for which level was highly increased in response to TDP-43 expression in glial cells. This was due to a reduction in the siRNA pathway, which is the major post-transcriptional silencing component of RTE in somatic tissue. Interestingly, reducing the glial expression of gypsy by RNA interference ameliorated the lifespan alteration induced by TDP-43 glial expression, strongly suggesting that loss of gypsy silencing was at least partially involved in TDP-43-induced toxicity [TBPH mutant [TBPH LOF share similar phenotypes.RNA-sequencing experiments have shown that pan-neuronal or pan-glial expression of TDP-43 induced upregulation of some retrotransposable element (RTE) in adult brain . Then, ttoxicity . This watoxicity . In additoxicity . AltogetH mutant , showingTBPH mutant heads showed that the levels of glutamic acid decarboxylase (Gad1) protein were reduced [TBPH mutant partially restored the defects in larval locomotion and the abnormal distribution of the post-synaptic GluRIIA glutamate receptors and Discs-large (Dlg) scaffolding protein. On the contrary, pan-neuronal silencing of Gad1 triggered larval locomotion defects and abnormal organization of GluRIIA and Dlg, as was observed in TBPH mutants. Interestingly, pan-glial expression of Gad1 completely rescued larval locomotion defects and abnormal GluRIIA distribution of TBPH mutants but not the Dlg one. Conversely, Gad1 silencing in glial cells induced locomotion defects and abnormal GluRIIA distribution but did not affect Dlg. Increased glutamate levels were found in the hemolymph of TBPH mutant or Gad1 knockdown larvae [Two-dimensional gel electrophoresis followed by mass spectrometry analyses on  reduced . This don larvae , suggestDrosophila pan-neuronal TDP-43 expression resulted in increased phosphorylation of eIF2α [Kim et al. found that of eIF2α . In respof eIF2α ,344. It of eIF2α , suggestof eIF2α . AltogetdFMRP [Drosophila ortholog of FMRP, which encodes a translational regulator and component of neuronal RNA granules [dFMRP enhanced and reduced TDP-43-induced eye degeneration and larval locomotor dysfunction, respectively [dFMRP GOF decreased the insoluble fraction of TDP-43, suggesting that excess of dFMRP may modify TDP-43 aggregation properties to restore translation [Using a candidate approach to search for RBPs that modulate TDP-43 toxicity, Coyne et al. identified dFMRP , the Drogranules . LOF andectively . In primnslation . Thus, tDrosophila, dAtx2 GOF aggravated the lifespan defects and eye degeneration induced by TDP-43. On the contrary, reducing dAtx2 dose rescued these phenotypes [Ataxin-2 is known to participate to SG assembly ,347. In enotypes . From thenotypes .PABP2, the Drosophila ortholog of PABPN1, enhanced the eye degeneration and the larval locomotor defect induced by TDP-43 GOF [PABP2 slightly increased the median lifespan of flies expressing TDP-43 in motoneurons [The poly(A)-binding protein nuclear 1 (PABPN1) was identified as a partner of TDP-43 . PABPN1 P-43 GOF . In primP-43 GOF . Thus, Poneurons , suggestDrosophila, both Parp knockdown and Parg overexpression suppressed the eye degeneration caused by TDP-43 GOF [Parp silencing also rescued age progressive climbing defects and reduced lifespan of pan-neuronal TDP-43 expressing flies [tankyrase (Tnks), which encodes a PARP, restored eye structure [Tnks knockdown rescued the lifespan defect induced upon pan-neuronal TDP-43 expression. Interestingly, the authors found PAR-binding motifs in the NLS region of TDP-43 that are required to localize TDP-43 to SGs [Tnks downregulation induced a redistribution of TDP-43 protein, with an increase in the nucleus and a decrease in the cytoplasm. This was likely due to an impairment of TDP-43 incorporation into SGs. Altogether, these studies showed that decreased PARylation is protective against TDP-43 toxicity.PolyADP ribosylation (PARylation) is a reversible PTM in which polymers of ADP-ribose are attached to proteins by PAR polymerase enzymes (PARPs) and removed by PAR glycohydrolases (PARG). PARylation was shown to regulate SG dynamics with a decreased level suppressing SG formation . In DrosP-43 GOF . Parp sing flies . A genettructure . Moreove3 to SGs . They alThe discovery that modulation of SG dynamics may modify the TDP-43-induced phenotypes opens the way to future studies targeting SGs for potential therapeutic strategy.Q331K mutant, but not wildtype or M337V mutant, was detected in cytoplasmic puncta when expressed in the developing eye [Q331K with doubletime, the fly ortholog of casein kinase Iε, strongly enhanced the degenerative eye phenotype induced by TDP-43Q331K but not the one induced by wildtype or M337V TDP-43 [doubletime, the Q331K mutant showed a higher level of S409/410 phosphorylation, which is detected in cytoplasmic inclusions of ALS patients [doubletime was co-expressed [Q331K was linked to abnormal phosphorylation and cytoplasmic aggregation. However, the reason why Q331K was more toxic compared to wildtype or M337V is still unclear. Nevertheless, this study suggests that targeting a specific kinase to modulate TDP-43 phosphorylation may be therapeutically interesting.Choksi et al. found that TDP-43ping eye . Co-exprV TDP-43 . Interespatients . In addixpressed . These dTBPH overexpression induced strong neurodegeneration in the eye that was completely abolished when GFP-12xQ/N was co-expressed [TBPH and GFP-12xQ/N were found co-localized in cytoplasmic aggregates in retinal cells. Biochemical fractionation experiments indicated that TBPH was mainly present in the soluble fraction upon eye expression, while it was predominantly present in the insoluble fraction when co-expressed with GFP-12xQ/N [TBPH is toxic and that aggregates have a protective effect, likely by sequestrating excess of TBPH. The same group showed that pan-neuronal expression of GFP-12xQ/N induced locomotion defects in mid-adult life [TBPH expression dropped during aging, which likely explains why TBPH sequestration by GFP-12xQ/N was deleterious during aging [Budini et al. developed a cellular model of TDP-43 aggregation by using a GFP-tagged construct composed of repetitions of the Q/N-rich C-terminal region of TDP-43 (GFP-12xQ/N) . In cellxpressed . In addiP-12xQ/N . Altogetult life . They alng aging .Nup50, Nup93, Nup98-96, Nup107, or Nup214 suppressed the TDP-43-mediated rough eye phenotype and rescued the larval locomotor phenotype induced by motoneuronal TDP-43 GOF [Nup50 rescued lifespan defects of motoneuronal TDP-43-expressing flies [Nup50 [Nup50 mutation was not due to a major nucleocytoplasmic redistribution of TDP-43 but rather to nucleocytoplasmic transport modulations. Thus, targeting some Nup proteins may have a beneficial effect on ALS disease.By using a proximity-dependent biotin identification approach, Chou et al. found that components of the nuclear pore complex and nucleocytoplasmic transport machinery were enriched in detergent-insoluble TDP-43 aggregates . They alP-43 GOF . This stng flies . Interess [Nup50 , suggestDrosophila retinal cells compared to control [Drosophila ortholog. When expressed in adult fly motoneurons, TDP-43 was found in cytoplasmic foci distributed in soma and neuronal processes. In contrast, co-expression of Clusterin restored the predominant nuclear localization of TDP-43 [The co-expression of TDP-43 with a GFP reporter of the unfolded protein response revealed that ER stress was elevated in  control . Then, t control , might mf TDP-43 . In addif TDP-43 . Thus, tDrosophila ortholog, dUBPY  resulted in higher amounts of insoluble and likely ubiquitinated TDP-43 in fly heads [dUBPY silencing enhanced the age-dependent eye degeneration induced by TDP-43 expression [M337V-induced eye degeneration was suppressed or enhanced by GOF and LOF of CG5445 gene, respectively [M337V, likely facilitating its degradation.The deubiquitinating enzyme UBPY (ubiquitin isopeptidase Y) was identified as an interactor of TDP-43 . The knoly heads . Additiopression . Anotherectively . CG5445 ectively . AccordiAltogether, these findings showed that modulating the unfolded protein response and ubiquitination might influence TDP-43 aggregation and toxicity, which may open the way for the discovery of potential therapeutic strategy.Drosophila ortholog of dual leucine kinase (DLK) [Wnd LOF and GOF extended and reduced lifespan, respectively. Effector kinases downstream of Wnd are p38 and JNK, two stress-activated kinases. The JNK pathway had a protective effect as the GOF of Basket, the JNK Drosophila ortholog, extended lifespan of flies expressing TDP-43 in motoneurons. Conversely, a reduced dose of Basket reduced lifespan. Contrary results were obtained with p38b, one the three p38 Drosophila orthologs, indicating that the p38 pathway was rather deleterious. This report also showed that oxidative stress and neuroinflammation were differentially modulated by Basket and p38b [A genetic screening, aiming to find modifiers of reduced lifespan induced by motoneuronal expression of TDP-43, identified the mitogen-activated protein kinase kinase kinase (MAP3K) Wallenda (Wnd), the se (DLK) . Wnd LOFand p38b , suggestDrosophila pan-glial TDP-43 increased the expression of the inflammatory genes Dorsal  [Ptp61F, the Drosophila ortholog of human Ptp1b encoding tyrosine phosphatase 1B, which is a ubiquitous enzyme anchored in the ER membrane, upregulated in neuroinflammatory conditions [Ptp61F silencing mitigated the age-dependent climbing defects and the reduced lifespan induced by glial GOF of TDP-43 [Ptp1b-dependent inflammatory response may be associated with TDP-43 toxicity.e, iNOS) . This wanditions . In addif TDP-43 . This stThus, inflammation processes may represent a target to counteract disease progression in ALS.TBPH mutant larvae, the phosphorylation levels of ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E (4E-BP1), two downstream effectors of mTORC1 [raptor mRNA was decreased in TPBH mutants. Raptor is an essential component of mTORC1 that regulates mTORC1 lysosomal localization. TBPH mutants also displayed an upregulation of some lysosomal and autophagic genes, as well as alteration in autophagosome–lysosome fusion [TBPH mutant aggravated larval locomotion. On the contrary, the mTOR agonist phosphatic acid ameliorated this phenotype. In both cases, the effects of rapamycin or phosphatic acid were dose-dependent [TBPH GOF [In f mTORC1 , were def mTORC1 . In addie fusion . Adminisependent . AnotherTBPH GOF . Altoget2+ channel localized in the endoplasmic reticulum, promoted cytoplasmic accumulation of TDP-43 and potentiated its recruitment to phagosomes [Itp-r83A, the only fly ortholog of ITPR1, increased the cytosolic fraction of TDP-43 in larval neurons and rescued the climbing defects and the reduced lifespan induced by motoneuronal expression of TDP-43 [A genome-wide RNAi screening identified the inositol-1,4,5-trisphosphate receptor type 1 (ITPR1) as a strong modulator of TDP-43 subcellular localization . The silagosomes . This suf TDP-43 . Thus, iDrosophila ortholog of human HSPB8, rescued the strong eye degenerative phenotype induced by TDP-43 mutated in its NLS domain (TDP-43NLSmut) but had no effect on the rough eye phenotype induced by wildtype TDP-43 [NLSmut but not wildtype was reduced when they were co-expressed. Conversely, RNAi-induced knockdown of HSP67Bc aggravated the TDP-43NLSmut-associated eye phenotype, which was correlated with a high accumulation of ubiquitinated proteins [NLSmut.Co-expression of HSP67Bc, the e TDP-43 . HSP67Bcproteins . Thus, HChd1, which encodes a nucleosome-remodeling enzyme required for heat-shock gene expression following stress [Chd1 co-expression [Berson et al. selected some genes related to various aspects of chromatin regulation and asked whether their downregulation may modify the TDP-43-induced rough eye phenotype . They fog stress . Upon hepression . Co-immupression . These dDrosophila hnRNPs may modify the degenerative phenotype induced by TBPH expression in the eye. In this way, Hrb27c, CG42458, Glo, Syncrip/Syp, and Hrp38 were identified as strong suppressors, while Hrb87F, Sm, Heph, and Rump were mild suppressors of eye degeneration [TBPH toxicity may be modulated by hnRNPs.Appocher et al. investigated whether RNAi-induced knockdown of neration . Thus, TClinical observations indicate that ALS patients have metabolic disturbances including body weight loss , hypermeSome alterations of lipid metabolism, such as a decrease in the carnitine shuttle and reduced lipid beta oxidation, were also found in larvae expressing TDP-43 (wildtype or G298S) in motoneurons . In addiAltogether, these studies showed that dietary intervention might be an interesting way to alleviate TDP-43 induced defects.TBPH have been identified in Drosophila TBPH mutant heads and at larval NMJs [futsch mRNA was highly enriched [TBPH construct was not able to restore the endogenous Futsch level as was observed with wildtype, indicating that the RNA-binding activity of TBPH was required to maintain futsch expression level [TBPH binds the futsch transcript to positively regulate its expression. The futsch mRNA was also enriched in immunoprecipitation experiments on flies expressing YFP-tagged TDP-43 in the eye [TBPH and TDP-43 may interact with futsch mRNA. When TDP-43 was expressed in motoneurons, the levels of futsch mRNA and protein were increased in the cell bodies, while they were decreased at the NMJs compared to control [futsch mRNA transport. In addition, polysome fractionation experiments indicated that TDP-43 induces the futsch mRNA to shift toward the untranslated fractions, strongly suggesting that TDP-43 represses the translation of fustch mRNA [futsch in TBPH GOF and no change in LOF condition [TBPH and TDP-43 directly bind the 5′-UTR of futsch mRNA [futsch mRNA region suggested that TBPH positively modulates the translational efficiency of futsch [One report found that the level of Futsch protein, a neuronal microtubule-binding protein homolog to human Microtubule associated protein 1B (MAP1B), was decreased in val NMJs . Immunopenriched . In addion level . Altoget the eye , indicat control , suggesttch mRNA . This isondition . It was sch mRNA , which cf futsch . It is nTBPH mutants, HDAC6 mRNA level was strongly decreased [HDAC6 mRNA level [TBPH bound HDAC6 mRNA [TBPH LOF might alter Brp acetylation and synaptic transmission.HDAC6 is a cytoplasmic deacetylase that plays an important role in the detection and degradation of ubiquitinated cellular aggregates . In TBPHecreased ,340, whiAC6 mRNA ,279. IntAC6 mRNA . Thus, icacophony transcripts was altered in TBPH mutant CNS [cacophony gene codes for a voltage-gated calcium channel localized at the active zone of NMJs that is required for neurotransmitter release. The level of cacophony protein was reduced in TBPH mutant larval NMJs, which have abnormal crawling behavior [cacophony fully rescues the locomotion behavior phenotype but not the pupal lethality associated with TBPH mutation, suggesting that the locomotion phenotype is mainly due to the lack of cacophony protein in motoneurons [cacophony may also be required in a few central neurons [TBPH antibody revealed the presence of cacophony transcripts, strongly suggesting that TPBH binds cacophony mRNA [cacophony observed in TBPH mutant decreased the cacophony protein expression level [TBPH is required for proper splicing of cacophony transcripts. Thus, these findings revealed that the splicing alteration of genes involved in synaptic transmission is a consequence of TBPH dysfunction, which might open new ways to better understand ALS pathogenesis.Transcriptome analysis revealed that the splicing of tant CNS . The cacbehavior . Interesoneurons . However neurons . RT-PCR ony mRNA . In addion level . AltogetDrosophila is a powerful genetic tool that has made major contributions to further our understanding of several neurodegenerative disorders, including ALS (Drosophila have allowed the identification of numerous ALS disease modifiers, paving the way for future studies in mammals. Genome-wide association studies and whole-exome/genome sequencing have accelerated the pace of identification of new ALS-associated genes, as illustrated for instance by the recent discovery of ALS-linked mutations in the kinesin family member 5A gene [Drosophila combined with their short lifespan will undoubtedly help to gain insights into the pathogenic mechanisms associated with these novel genes or variants.ding ALS . Further 5A gene ,372. MorDrosophila.Despite the well-recognized motoneuronal death in ALS, motoneuron neighboring cells including astrocytes, oligodendrocytes, and interneurons degenerate before motoneuron loss , stronglDrosophila will certainly help to identify and characterize novel molecular and cellular mechanisms involved in this disease, giving hope for the development of therapeutic strategies.Despite intensive research over the past decades, no efficient therapy is currently available for ALS.


In this paper, the hybridized localized surface plasmon resonances (LSPRs) of a periodic assembly of graphene-wrapped nanoparticles are used to design a nanoparticle assisted optical absorber. Bandwidth enhancement of this structure via providing multiple types of plasmonic resonances in the associated unit cell using two densely packed crossly stacked graphene strips is proposed. The designed graphene strips support fundamental propagating surface plasmons on the ribbons, and gap plasmons in the cavity constructed by the adjacent sections. Graphene strips exhibit a hyperbolic dispersion region in the operating spectrum and assist in the bandwidth enhancement. Moreover, since the nanoparticles are deposited on the top strips, real-time biasing of them can be easily conducted by exciting the surface plasmons of the strip without the necessity to electrically connect the adjacent nanoparticles. The overall dynamic bandwidth of the structure, using a two-state biasing scheme, covers the frequencies of 18.16–40.47 THz with 90% efficiency. Due to the symmetry of the structure, the device performs similarly for both transverse electric (TE) and transverse magnetic (TM) waves and it has a high broadband absorption rate regarding different incident angles up to 40°. Due to the presence of 2D graphene material and also using hollow spherical particles, our proposed absorber is also lightweight and it is suitable for novel compact optoelectronic devices due to its sub-wavelength dimensions. As an instance, simple cubic patterns of gold cylindrical and spherical particles have been proposed to convert the laser energy into heat in non-destructive testing and medical diagnostic applications3. Moreover, an optical meta-surface constructed by silver ellipsoidal particles behaves like a resistive sheet and by engineering its surface impedance, a Salisbury absorber can be designed4. These narrowband absorbers can be possibly used in light filters or digital sensors5.Nanoparticle assisted optical absorber design is a well-known approach for manipulating the interaction between the electromagnetic wave and matter at infrared and optical frequencies6. Graphene, a two-dimensional nanomaterial, is highly resistant to oxygen. Besides, due to being chemically inert and strongly transparent to light, it is a promising candidate in various novel optical devices7. Considering the graphene-wrapped structures, free-standing spherical nanoparticles behave as a reconfigurable reflector away from their plasmonic resonances8. Moreover, by dispersion engineering of the resonance channels provided with the plasmonic resonances of a double graphene layer spherical particle, a superscatterer can be designed9. Enhanced energy transfer and giant near field enhancement are two other examples exhibiting the wide range of applications provided by localized plasmons in graphene-coated particles11.An inherent drawback of metal-based nanoparticle assisted absorbers is the formation of an oxide shell on the surface of the nanoparticle due to contact with air or moisture. This layer may significantly affect the scattering pattern and should be taken into account in practical applications12. Focusing on graphene-wrapped structures, enhanced absorption is reported in the sub-wavelength graphene-coated wires13. Moreover, by preparing the graphene-coated particles on top of an optical mirror, the plasmonic resonances result in the narrow-band perfect tunable optical absorption, originating from the large extinction cross-section of an isolated particle concerning its geometrical cross-section15.Specifically, graphene-based perfect metamaterial absorbers have attracted a surge of research interest due to the extraordinary electronic and photonic properties of graphene material19. For instance, broadband absorption in graphene-wrapped fractal oligomers is the result of plasmonic resonances of various orders arising from plasmonic hybridization in the top, bottom, and lateral surfaces19, revealing that the combination of the two of the aforementioned methods is used. Bandwidth enhancement of optical absorption in graphene-based devices can also be achieved by combining graphene with various photonic structures, such as dielectric waveguides, photonic crystals, plasmonic metamaterials, dielectric metamaterials, integrated microcavities, and other types of resonant structures21. Hyperbolic metamaterials (HMM), being anisotropic artificial materials, are other alternatives that have been widely used in manipulating optical absorption23. In this regard, broadband absorption using tapered HMM array is experimentally realized in the visible and infrared ranges24. In another research, ultra-broadband light absorption based on photonic topological transitions in HMMs is proposed25. Moreover, a polarization-dependent broadband infrared absorption inside the hyperbolic frequency region is attained using α-phase molybdenum trioxide (MoO3) nanostructures26.Generally, bandwidth enhancement in the resonant structures can be achieved by providing various closely packed resonances, exploiting multi-resonance unit cells, multi-layered structures, and multiple-order resonances27. Stacked graphene-dielectric pairs can be used to realize gate tunable hyperbolic metamaterials to be possibly used as a super-absorber28. Hyperbolic meta-surfaces based on nanostructured van der Waals materials provide opportunities for the realization of subwavelength-scale structures29. Hexagonal boron nitride (hBN) and borophene are examples of the two-dimensional Van der Waals natural hyperbolic materials, respectively used in the design of the multi-band and single band absorbers31, and both of them are lacking the gate tunability feature. The hyperbolic plasmons of densely packed graphene strips enable voltage reconfigurable flatten optics in a broad bandwidth32 and it is of interest in this research.Hyperbolic metamaterials are commonly designed by stacking alternating metal and dielectric layers or using a lattice of metallic nanowires embedded in a dielectric matrixTo design a broadband absorber, we have exploited the multi-resonance, multi-layered, and multi-ordered schemes simultaneously by using hollow graphene-wrapped particles on top of two densely packed crossly stacked graphene strips. It is shown that the hybridized localized surface plasmon resonances in the graphene-coated spherical particles result in the excitation of quadrupole modes that cover the high frequencies of the desired spectrum. These resonances are manipulated with the propagating surface plasmons and gap plasmons of densely packed dual graphene ribbons to enhance the operating bandwidth. The reason for choosing the densely packed strips as the other resonating element of the unit cell is its capability to support hyperbolic plasmons.33. Grating-based graphene structures can be simply biased with a cathode in the form of a bridge34 and real-time biasing of each ribbon is also feasible by using field-programmable gate array (FPGA) hardware35. Therefore, in the proposed design, the LSPRs in an assembly of isolated spherical nanoparticles are excited by residing them on top of the graphene strips. Note that exciting the localized surface plasmons via propagating surface plasmons is a well-known approach that was proposed for the metallic nanoparticles before36 and our design benefits the same strategy for a graphene-based structure.Our design offers polarization-insensitive optical response due to the crossly stacked strips and it is lightweight due to the use of 2D graphene material and hollow spherical particles. Another feature of our proposed structure is offering a simple biasing scheme. In general, graphene-based devices with discontinuous elements suffer from the fabrication complexity due to the requirement for properly designed biasing networks37. Moreover, as a result of the van der Waals force, graphene can be wrapped around particles of various shapes and sizes38. For instance, graphene oxide wrapped gold nanoparticles have been fabricated by a one-step synthesis39. Densely packed graphene strips can be fabricated by electron beam lithography40. The plasmonic grating with gold nanoparticles resided on its surface is realized by respectively fabricating the sections with the colloidal self-assembly and self-assembly techniques41. Alternatively, electrohydrodynamic (EHD)-jet printing is proposed to attain a nanoparticle assembly on the electrostatically attractive surface42. Considering the above-mentioned techniques, our proposed structure is also experimentally realizable with current nanofabrication technology.As a final comment, graphene material can be prepared by chemical vapor deposition (CVD) or by epitaxial growth on silicon carbide or metals44. The analytical investigation of our proposed structure is complicated due to the tensorial surface conductivity required for the appropriate modeling of the equivalent surface conductivity of graphene strips45. Therefore, we will focus on the numerical analysis of this structure.The paper is organized as follows. Initially, the optical performance of densely packed graphene strips and graphene-coated spherical particles are disclosed. Later, dual graphene ribbons are hybridized with the graphene-based meta-surface constructed by the spherical particles for bandwidth enhancement. Due to the contact of particles and top strips, the dynamic biasing network can be easily implemented through optical bridges, connecting the strips. It is worth noting that a typical approach to design the metamaterial plasmonic light trapping devices is solving Maxwell’s equations via computational electromagnetics such as the characteristic matrix method, finite element method, and circuit design, to name a fewIn this section, the optical performance of densely packed graphene strips and graphene-wrapped spherical particles are discussed. These two elements will be used in the unit cell of our proposed broadband absorber in the next section. Matlab-based programs are used to investigate the effective surface conductivity of graphene-based strips and the extinction efficiency of graphene-coated nanoparticles. The results of these subsections will aid to opt suitable initial values for the optical and geometrical parameters.W and periodicity of L. The strips can be biased through a bridge using electrostatic bias voltage v1. The influence of the thin bridge is neglected throughout the paper. This structure behaves as a uniaxial meta-surface and it supports a hyperbolic dispersion region enabling the flattened optics40. To benefit the hybridization of the hyperbolic plasmons with the LSPRs, it is essential to extract the hyperbolic dispersion spectrum of the graphene strips considering various geometrical and optical parameters. The isotropic surface conductivity of the continuous graphene sheet 46:T are respectively the electron charge, Boltzmann’s constant, reduced Planck’s constant, and temperature. Moreover, τ = 32.9 fs)47, throughout this paper. The applied bias voltage alters the graphene chemical potential μc and these two parameters can be related via approximate analytical equations48. Note that the chemical potential as high as 2 eV can be obtained experimentally49. The elements of the uniaxial surface conductivity of the densely packed graphene strips are50:εeff is the effective permittivity of the substrate, and G is the gap distance between two adjacent strips. The hyperbolic dispersion region covers the spectrum in which the imaginary parts of the diagonal surface conductivities have opposite signs. To investigate this condition, Fig. W = 220 nm, L = 240 nm, and considering various chemical potentials. The symbol 51. Based on Fig. 50. The hyperbolic region can be further manipulated by varying the parameter G. In Fig. L = 240 nm, and the chemical potential of μc1 = 1 eV. Increased gap distances lead to the blue shift of the transition frequency.Let us consider the free-standing densely packed 2D graphene strips of Fig. ε1 = 1) with the radii R ranging from 70 to 100 nm, the relaxation time τ  = 32.9 fs, and μc = 0.95 eV is illustrated in Fig. 52. The normalization factor is the geometrical cross-section of the spheres. In general, increasing the sphere radius red-shifts the resonance frequency. Moreover, the higher-order modes are not strongly excited in the isolated spheres, but, they play a crucial role in the performance of the assemblies of these particles, as will be discussed later.To get some insight into the localized surface plasmon resonances of the graphene-coated spherical particles, an isolated particle under plane wave illumination, as shown in the inset of Fig. R = 100 nm and optical parameters of τ = 32.9 fs, and μc1 = 0.95 eV for different values of the periodicity, as in Fig. 53, is attained. The impact of periodicity in this interval is to set the frequency peak. Note that although particles with sub-nm gap distances are experimentally realizable54, we have chosen the minimum distance as 20 nm to prevent the proximity and non-local effects55. Note that the value of the chemical potential matches with the optimized value of the next section in the final design.Regarding the optical absorption of free-standing sphere array with the radii 47, simple expressions to evaluate the complex frequencies characterizing the extinction resonances of a single graphene sphere is provided by calculating the poles of the modified Mie–Lorenz coefficients, where its real part gives the peak center and its imaginary part denote its full width at half maximum (FWHM), approximately38. Noting that sparse arrays resemble the isolated spheres, once the particles are arranged in an array, a spectral broadening is evident. Both redshift and spectral broadening are produced by coupling effects between spheres in the array. In a dense 2D array of plasmonic sub-wavelength spheres, as the sphere radius increases or as the lattice constant decreases, i.e., as the coverage increases, the parallel mode resonance is shifted towards lower frequencies and the normal mode resonance to higher frequencies56. Since the normal-incidence for the illuminating plane wave is considered here, only parallel mode is present in our simulations and its performance is in agreement with56. Note that this figure is produced using CST 2017 software package and the simulation setup will be introduced in the next section in detail.In Ref.47, the associated localized surface plasmon resonances will lie on the hyperbolic dispersion region of the densely packed strips leading to an efficient wideband enhanced absorption.Based on the results of the previous two subsections, by wisely choosing the optical and geometrical parameters of the graphene-wrapped spherical particlesIn this section, broadband absorber design using graphene-based grating-coupled nanoparticles will be presented. The unit cell consists of a graphene-coated hollow spherical particle resided on top of the two densely packed crossly stacked graphene strips. The necessity of each element for the broadband response is clarified and excited surface plasmons are exhibited by providing the spatial distribution of the electric field. Finally, various parametric studies are conducted to discuss the influence of the geometrical parameters on broadband optical absorption.W = 220 nm and gap distance of G = 20 nm is illustrated in Fig. R = 100 nm have prepared on the top of the 2D hyperbolic meta-surface. Therefore, by exciting the propagating surface plasmons of the strips, the localized surface plasmons of the particles are excited due to electrical connection. The relative distance between the two strips is d = 100 nm and the lower one is rotated 90° concerning the other one. The reason for this modification relies on the fact that even-layered crossly stacked anisotropic 2D material nano-structures support complementarily excited surface plasmon resonances in the two lattice directions and are capable of polarization-independent absorption57. For simplicity, the geometrical parameters of both strips are considered to be identical. The supporting substrate is a dielectric with the height of h = 1900 nm and relative permittivity of εd = 258. There is also a metallic mirror beneath the substrate to contribute to the absorption enhancement by suppressing the transmitted wave59. This layer is constructed by gold (Au) material with the dispersive constitutive parameters extracted from60. This layer is also beneficial in the electrostatic biasing of the device as a reference plane61. The 3D view of the absorber is exhibited in Fig. 34. The subsequent results are provided with the unit cell analysis of the frequency domain solver of CST 2017 commercial software package. In all simulations, the first TE and TM modes are considered to be excited in the Floquet ports, unless otherwise is stated.In the following paragraphs, the previously introduced 2D hyperbolic meta-surface will be used as a substrate for the broadband absorber design. The unit cell of our proposed wideband absorber which is constructed by two stacked densely packed graphene strips with the width of μc1, μc2) (eV) which are associated with the bias voltages  (V) is illustrated in Fig. 58. The advantage of our proposed structure is using a much smaller unit cell. The periodicity in Ref.58 is 10 μm while in our design it is 240 nm. Similar operating wide bandwidths can be also found in Refs.63, respectively designed with four-ring elements and tapered patch antennas. Note that wideband absorbers based on graphene sheet or pattern are mainly designed in the terahertz frequencies and lower66. The dynamic bandwidth of the proposed absorber will be compared with graphene-based particle assisted absorbers in the last section. It should be emphasized that loosely coupled graphene strips behave as RLC resonators and enables the optical absorption at resonance67. In the presented simulations, graphene isotropic local surface conductivity is applied to the densely packed strips. The procedure of extracting the anisotropic effective surface conductivity of hyperbolic meta-surfaces using commercial software packages can be found in68.The absorption spectrums of our proposed structure for a set of chemical potentials  nanoparticles, they should be mixed by ultrasonic dispersion in the deionized water, then dried by vacuum freeze-drying process. After annealing the resulted powder in argon gas, the SiO2 template can be removed by immersing 20% hydrofluoric acid (HF) solution with stirring70.Another outstanding feature of our proposed absorber is using the graphene-wrapped hollow particles in the design. Core material impacts the absorption amount of graphene-wrapped spheres since its decrease is equivalent to the decrease in the geometrical size of the system resulting in blue shift and enhancement of absorption efficiency47. For the sake of clarity, the surface charge distributions which are proportional to 47 for the quadrupole modes (l = 2) of various degrees . The symbol n values differ from their positive counterparts only by a rotation. It should be noted that higher-order plasmonic resonances are also capable of efficient energy absorption71 and they are excited due to the hybridization72.To further investigate the performance, the electric field distributions at three frequencies are illustrated in Fig. mentclass2pt{minim73. Moreover, gap plasmons occurring between a given sphere and the graphene strip set below, enable plasmonic coupling in the form of gap plasmons with increased absorption. Gap plasmons exhibit the concentration and thus the absorption of the confined fields due to the large effective index75. These resonances appear only for the sufficiently narrow inter-element gaps in which highly coupled plasmons result in a metal–insulator–metal (MIM) like performance supporting a cavity mode or plasmonic standing wave, and they are characterized by strong absorption of the incident light77. Note that excitation of gap plasmons have been reported using a variety of structures including but not limited to a Bottle-like narrowband absorber, MIM structures with nanometer-sized spacers, deep metallic grating with narrow slits, and paired nanowire waveguides81.The excited plasmons on the sub-wavelength strip gaps are illustrated in Fig. Ey in Fig. Ez in Fig. 83. Our proposed structure can be considered as a substrate mediated plasmonic hybridization technique similar to the graphene decorated silver nanoparticles, metallic nanoparticle-dimer on a mirror, and nanoparticle/graphene/film combinations86.Another efficient resonance for the absorption is the propagating surface plasmons in the stacked strips. This particular mode in the strips shows an antinode (max/min) at the center of the strip for θ is varied from 0° to 80° for TE and TM polarizations. The simulation results, illustrated in Fig. 87. The minor difference in the performance of TE and TM modes is attributed to residing graphene-wrapped particles on the top strip.To investigate the influence of the polarization and incident angle of the electromagnetic source on the amount of absorption, the incident angle G is varied from 30 to 80 nm. In all the simulations the strip widths are decreased to maintain the previous value of the periodicity. Based on the results of Fig. The impact of the geometrical parameters on the optical performance of the absorber is investigated in Fig. G = 80 nm is included in Fig. Ez in Fig. W = 220 nm (corresponding to G = 20 nm) in Fig. G, the absorption band coincides with the hyperbolic regime of the graphene meta-sheet, as shown in Fig. R is swept from 60 to 90 nm in Fig. The electric field distributions at two resonance peaks for d = 80–400 nm, the performance is slightly changed, indicating that the inter-coupling of surface plasmons in graphene sheets is a neglecting mechanism for absorption, enabling a robust performance against fabrication tolerance. This performance is expected due to the extreme light confinement in the graphene-based densely packed strips40. Similarly, the local nature of surface plasmons has resulted in the periodicity independent behavior in another graphene-based absorber based on magnetic dipole resonances88. This result can be further confirmed by comparing Fig. d + h = 2 μm.Figure 89, the maximum attainable bandwidth using a single layer of graphene-coated particles is around 7.13 THz which can be extended to 13.96 THz by stacking double layers of such particles with optimized parameters. The present design has a single layer of particles and its substrate thickness is around half of Ref.89 while providing 22.31 THz of dynamic bandwidth. This design offers the opportunity to the dynamic tuning of the chemical potential, as well. On the other hand, the dynamic tuning of the absorbers in Ref.89 are not practically feasible due to the isolation of the particles. The bandwidth can be further improved by engineering particle size to excite other plasmonic resonances19.Finally, note that the chemical potential for the wideband performance in Fig. By simultaneously exciting the localized surface plasmon resonances of graphene-coated nanoparticles and the propagating surface plasmons of stacked densely packed graphene strips, a wideband optical absorber can be realized. Bandwidth enhancements can be interpreted based on the anisotropic nature of tightly coupled graphene strips which behave as a 2D hyperbolic material in the desired operating bandwidth. The narrow gaps between the strips result in the excitation of the gap plasmons as another absorption enhancement mechanism. Moreover, once these anisotropic layers are stacked in the cross-shaped form, they provide a polarization-independent absorption. The proposed structure offers a simple approach for practical biasing of the graphene-based nanoparticles deposited on the strips. The real-time plasmonic performance tuning is feasible to further enhance the dynamic bandwidth of the structure. The wideband absorption bandwidth of our proposed structure is maintained for the oblique angles up to around 40° and it is lightweight and sub-wavelength.


Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies. HPV is implicated in more than 70% of head, neck, and throat cancers, the occurrence of which has been increasing steadily, specifically in a younger group of non-smoking 40–50 year old white men5. Although there are over one hundred known genotypes, HPV16 is associated with 50% of all cervical cancers and 90% of all other HPV-containing anogenital and oropharyngeal malignancies10. We used HPV16 in this study due to its disproportionate contribution to human disease.Human papillomaviruses (HPVs) cause epithelial tumors and are the etiologic agents of numerous anogenital and oropharyngeal cancers13. Pseudovirus is comprised of both structural proteins (L1/L2) with plasmid DNA packaged as a mock genome15. Quasivirus is similarly comprised of both structural proteins (L1/L2), but packaged with a cottontail rabbit papillomavirus genome (CRPV) to assemble a structurally complete L1/L2 capsid that is infectious16. All these different HPV VLPs preserve the main attributes of the native capsid structure and have been used successfully for vaccine development and for studies of antigenicity, receptor usage, entry mechanisms, and structural analyses. Quasiviruses were used throughout the work described here.HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high-titer virus preparations for research problematic. Consequently, alternative HPV production methods of virus-like particles (VLPs) have been developed within the HPV community for virological and structural studies. Capsid protein only VLPs can be composed of the major structural protein (L1) alone, or of the major and minor capsid proteins (L1/L2) and are not infectious since they are devoid of viral genomes18. Five copies of L1 intertwine to form each of the 72 capsomers that make up the T = 7d icosahedron. The L2 structure is unknown and its capsid incorporation is unclear. L1 takes the form of the ubiquitous jellyroll of anti-parallel beta strands (BIDG and CHEF), which are connected by flexible loops that extend outward from the surface of the capsomer and constitute the majority of the L1 hypervariable regions. Pentavalent capsomers lie on the icosahedral fivefold axis and are surrounded by five neighboring capsomers. The remaining sixty hexavalent capsomers are each bordered by six capsomers. To form the icosahedral capsid the pentavalent and hexavalent environments confer quasi-equivalent conformations of L1. The asymmetric unit is comprised of six L1s, five of which make up a single hexavalent capsomer and one L1 that contributes to a pentavalent capsomer , to assess capsid flexibility on a per particle basis. Our high resolution map provided the most reliable structure of L1 to date, including corrections to the previous model. The structure also revealed for the first time unambiguous L2 density adjacent to a conserved L1 loop. Assessment of capsid flexibility revealed dynamic capsids with imperfect icosahedral symmetry. This continuous heterogeneity is the likely cause of limited resolution of HPV structure in previous studies and may play a role in crucial biological processes. The work describes methods to achieve high resolution that will lay the groundwork for future structural studies of papillomavirus and polyomavirus.22. Using a standard processing pipeline , a suite of programs inspired by the Localized Reconstruction approachops Fig. C,D. The ops Fig. E,F were Notably, resolution did not improve beyond 3.1 Å, despite exceptional subparticle number , which suggested the presence of additional heterogeneity. However, multiple attempts to 3D classify the subparticles, both before and after local refinement, failed to produce structurally distinct classes. This suggested that the signal-to-noise ratio (SNR) is the limiting factor in achieving truly atomic resolution. Optimized and iterative subtraction of neighboring subparticles might improve the resolution further, but such an experiment would require new software development and achieve modest improvements if successful.22. However, as the model contained the asymmetric unit, it was used to initiate the build. Each L1 chain of the asymmetric unit was initially built into the corresponding capsomer subvolume density. The pentavalent capsomer is comprised of chain A with connecting arms from chain F, whereas, the hexavalent capsomer is comprised of chains B–F with the connecting arms from chains A–E  was accurate to only modest resolution since it was generated from a 4.3 Å cryo EM density map.) allowed us to correct the 36 residues making up the C-terminal arm extensions (402–439)22. This map clearly resolved density corresponding to the Lys475 to Phe480 loop that was previously incorrectly assigned. N-terminal residues starting from Met 1, which had previously been unresolved, were now assigned for most of the chains in the asymmetric unit. Overall, the density in this map allowed clear assignment of residues and corrected the HPV16 L1 structure within the context of the icosahedral capsid.After refining the structures in each of the subvolumes, the six L1 chains were assembled into the asymmetric unit. This pdb was then validated against the recombined map S. Table  and presThe pentavalent and hexavalent L1 capsomers share a common core architecture but have different connecting arm structures (residues 402–439). The connecting arm extends from the base of the donor capsomer, looping into the neighboring capsomer to make a disulfide bond between Cys428 of the connecting arm and Cys175 of the neighboring capsomer, and then returns to the donor above the initial extension.Capsomers superimposed with a c-alpha RMSD of 2.75 Å Fig. A; howeveThe fit of these connecting arms revealed that the same sequence was found in different conformations. The connecting arms were resolved to a more modest resolution compared to the capsomer core. Specifically, the surface exposed region of the arms from His431 to Asp439 was found to be poorly resolved in each chain, even though the connecting arms are in varying conformations. The most compact conformation was in L1 chains A and F, whereas in chains D and E the conformation was the most extended, with chains B and C exhibiting an intermediate structure  there was inadequate SNR for successful classification (2) L2 may have long disordered stretches between the locations that we were able to resolve.The original location of each extracted subvolume was recorded using ISECC S. Table . Hexaval22.The diameter of individual HPV particles was calculated using the capsomer metadata recorded by ISECC. Particle diameter was defined as the x,y distance along the icosahedral fivefold axis between pentavalent capsomers on opposite sides of the capsid. In order to minimize unmeasurable distances out of the image plane, this analysis was necessarily limited to particles where the polar-opposite fivefold capsomers lay within the same z plane, allowing a 5% radial tolerance 12.9 Å) from z = 0 Fig. ” section2.9 Å froIn addition to the improved resolution, local subvolume refinement allowed a determination of capsomer centers and orientations for the first time. The movement of capsomers relative to their icosahedrally-forced  parameters suggests flexibility. The corrected capsomer centers were used to evaluate the relative motions of capsomers within the imperfectly icosahedral HPV capsids. Capsomer movement was evaluated for all hexavalent subparticles within 25 Å of x,y distance from the particle center. This criterion was selected to maximize lateral, in-plane movement of capsomers and limit out-of-plane motion, reflected in defocus, that cannot be recovered using local refinement. An additional filter was used to exclude any capsomer whose icosahedrally-derived center was within 15 Å x,y distance of another, such as capsomers on the front and back of the capsid, as the identities of such capsomers could be confused during local refinement see “” sectionFlexibility analysis suggested that variation in capsid diameter is due to variable contraction or expansion of the flexible C-terminal arms linking the capsomers, which in turn move as rigid bodies. This is consistent with the poor resolution of residues 431–439, found in the connecting arms, that was seen in all chains of the asymmetric unit in both the icosahedrally refined and locally refined maps. However, we saw no evidence suggesting that any one capsomer-to-capsomer linkage is more or less flexible than another. This observation is surprising considering some capsomers are paired by donation of a single arm (chain B:Null), whereas others are linked by bidirectional arm exchange  Fig. B.22. This resolution limitation cannot be directly attributed to the use of VLPs with unauthentic genome (quasi or pseudovirus), as studies with native papillomavirus have encountered the same resolution barrier, suggesting papillomaviruses are inherently flexible44. Future studies with native virus will allow for analysis of the impact authentic genome may have on the overall flexibility of the capsid. Here we show that even with recent improvements in cryo EM hardware and software for whole particle sorting, classification, and averaging, the resolution has not improved. However, we found that HPV responded well to a subparticle refinement approach that isolated individual capsomers for local refinement. Thus, we were able to improve the resolution from 4.5 to 3.1 Å. The maps of individual capsomers were subsequently recombined to create a 3.1 Å resolution model of the complete icosahedral virus capsid. The high resolution and the structure of the L1 asymmetric unit allowed us to better understand the different conformations of the connecting arms that link capsomers. Although each L1 chain is chemically identical, each 37 amino acid loop makes a different connection to its neighboring capsomer, due to quasi-equivalence. When looking at the six chains of the asymmetric unit, the low RMSD of the L1 capsomer cores, compared to the higher RMSD of the connecting arms, suggests that the L1 core secondary structure is conserved whereas the arms have different conformations. One thing that is consistent in all chains of L1 within the asymmetric unit is the modest resolution of His431 to Asp439, which are solvent exposed on the surface of the virus, and the poor resolution is likely attributed to those nine amino acids being a source of flexibility.Previous icosahedral reconstructions of papillomaviruses never surpassed ~ 4.3 Å resolutionThe near atomic resolution of the map allowed us to identify protein-like density unfilled by L1, which corresponded to the minor capsid protein, L2. In our map we can see clear separation between the L1 chain and regions of putative L2, which have likely been stabilized by their interactions with L1. Notably, there is a high sequence conservation of the flanking L1 residues that interact with L2 in these regions. However, the putative L2 density is not as strong as L1 in either location although the magnitude of putative L2 density is stronger in the pentavalent than the hexavalent capsomer  between capsomers22. Because subparticle refinement was able to determine the true center of each capsomer, we were able to use these new values to determine the actual diameter of each capsid on a per particle basis using ISECC_local_motions.py script  from their idealized values, without bias in the magnitude or direction of capsomer movement Fig. . This fl. in hepatitis B virus45. We propose a model in which the HPV capsid is dynamic and in a state of constant flexing. This model is supported by the composition of stable beta-jellyroll capsomer cores with flexibility stemming from the connections between these capsomers. The classification of different diameter particles likely failed because the intercapsomer flexing is global and variable, not by symmetrical coordinated movements. Thus, even for particles with the same diameter, the capsids have different configurations due to the flexing between capsomers that creates imperfect icosahedral symmetry. These elements of heterogeneity include a continuous range of different diameters due to deviations of capsomer positions from a perfect icosahedral grid, which has likely limited the resolution attained through whole-capsid refinement.Although dynamics information cannot be conclusively derived from a vitrified population, each particle may represent a state within an equilibrium of particles in solution. These states, described for the first time by ISECC, provide a source of experimental capsid dynamics data that could contribute to whole-capsid molecular dynamics simulations such as described by Hadden et alThe observed dynamics may be a natural consequence of the architecture of the T = 7d icosahedral capsid that requires L1 to exist in hexavalent and pentavalent environments. However, there are a multitude of possible benefits to a dynamic capsid that allows reversible expansion and contraction. This flexing could serve as a defense mechanism to hide or alter antibody binding sites, inhibiting recognition and preventing binding and neutralization. Capsid flexibility also has the potential to make HPV more resilient to environmental factors such as pH, temperature, or desiccation. Mechanistically, dynamics are certainly necessary for the conformational changes that are known to occur during host cell entry. Lastly, in the final stages of the virus life cycle, mature virions pack within the nucleus in a paracrystalline array of compact capsids that are significantly smaller diameter than when observed after lysis in suspension. Thus, capsid flexibility is an intriguing and essential factor to papillomavirus function..46). The design of stable antigenic epitope-expressing HPV particles will be improved with the cryo EM structural analyses presented in this study, and lead to in silico rational design and selection of stable VLP vectors for vaccines and gene-therapy deliverables.Until now, high resolution structures of HPV have been unattainable. By using subparticle refinement approaches we demonstrated that high resolution is achievable. The new, more accurate L1 structure revealed putative L2 density flanking the conserved 306-loop of L1. We also found that the HPV capsids are globally flexible with capsomers moving as rigid bodies. These findings provide a framework for continuing structural, biochemical, genetic, and biophysical studies of HPV. These new observations on capsid flexibilities containing rigid capsomer units paves the way for design of HPV capsid platforms that can deliver foreign antigenic epitopes engineered for the development of second-stage vaccines  was transfected together with linear CRPV/SV40ori DNA into 293TT cells and prepared as described previously50. HPV16 was allowed to mature and then pelleted by centrifugation. The centrifuged pellet was resuspended in 1 M NaCl and 0.2 M Tris (pH 7.4). After CsCl gradient purification, the lower band was collected. The lower band was then added to CsCl for another round for ultracentrifugation to separate full from empty virus particles. The lower band was collected, concentrated, and buffer exchanged using a 100KDa cutoff spin column as described previously23. The concentrated HPV16 quasivirus particles were applied to 300 mesh carbon coated copper grids and stained with 2% phosphotungstic acid. The sample was analyzed for integrity and concentration on an FEI Tecnai G2 Spirit BioTwin transmission electron microscope.HPV16 quasivirus containing L1 and L2 proteins and encapsidating a CRPV genome having the SV40 origin of replication was prepared as described previouslyhttps://www.huck.psu.edu/core-facilities/cryo-electron-microscopy-facility/instrumentation/fei-titan-krios). 3.5 µl of the purified virus sample was pipetted onto a glow-discharged R2/1 Quantifoil grid , blotted for 2.5 s, and plunge-froze in liquid ethane using a Vitrobot Mark IV . Vitrified grids were imaged with the use of a Titan Krios G3  under automated control of the FEI EPU software. An atlas image was taken at 165 × magnification, and suitable areas were selected for imaging on the FEI Falcon 3EC direct electron detector. The microscope was operated at 300 kV with a 70 μm condenser aperture and a 100 μm objective aperture. Magnification was set at 59,000 × yielding a calibrated pixel size of 1.1 Å. Four, nonoverlapping exposers were acquired per each 2-um-diameter hole of the grid with the beam in parallel mode, for an overall collection of 10,143 micrographs. The total dose per exposure was set to 60 e−/Å2 (Supp. Table The HPV16 sample was assessed for purity and concentration before vitrification for cryo-EM data collection on the Penn State Titan Krios (51. The micrographs underwent full frame motion correction and CTF estimation (CTFFIND4)52. Micrographs were curated and sorted to reject micrographs with crystalline ice. Particles were picked using 2D templates from 840 particles. Local motion correction was performed on the particle stack and the CTF estimated micrographs. The particles underwent 2D and 3D classification in order to select only for particles containing internal (genome) density. The resulting particles then went into a homogenous refinement. The final resolution was determined by gold standard FSC threshold of 0.143.Icosahedral refinement was performed in cryoSPARC53. The subparticles were then processed in RELION v3.153.ISECC_subparticle_extract was used after icosahedral refinement to divide each particle image into subparticles. Both hexavalent and pentavalent capsomer subparticle were separately created. An initial model was made for each subparticle type using relion_reconstruct with 10,000 subparticlesPentavalent subparticles were locally refined with C5 symmetry whereas hexavalent C1 was used to refine the pentavalent subparticles. Each subparticle dataset was refined with a spherical mask of 200 Å applied to focus on the capsomer and adjoining arms. The final resolutions were determined by gold standard FSC with a threshold of 0.143 in RELION post-process. Local resolution maps were generated using RELIONs own software.40.Postprocessed subparticle maps were recombined into a complete capsid using ISECC_recombine. This procedure is similar to the recombination process for subparticles in Block Based Reconstruction and LocalRecBriefly, ISECC_recombine loads the subparticle maps into a numpy array and both shifts and rotates the maps to their locations in an idealized icosahedron, using regular grid interpolation in real space. This interpolation scheme allows merging of the subparticle-refined models into a single asymmetric unit.22.The previously solved L1 structure (PDB: 5KEP) was used to initiate the build55. The L1 protein structure from HPV16 (PDB: 5KEP) was used as an initial model. The asymmetric unit PDB was used to create a protein structure for the pentavalent and hexavalent capsomers. The hexavalent capsomer was made from chains B–F of the existing PDB and the pentavalent capsomer model was made from 5 copies of chain A from the asymmetric unit. These models were fit into the electron density map of the pentavalent and hexavalent capsomers independently in Chimera56. The protein structure of L1 was then refined in real space against the cryo-EM electron density map in Phenix with geometry and secondary structural restraints54. The structure was visually inspected and manually refined in Coot and validated using MolProbity57 , --roi , --subpart_box , --supersym (symmetry of the whole particle: I1 or I2).ISECC introduces several new metadata labels to the RELION 3.1 star file to enable correlated analysis of subparticles after local refinement or classification. For the idealized icosahedron, each vertex is given a designation, rlnCustomVertexGroup, containing the symmetry axis and an integer  SFig . If the Metadata label rlnCustomOriginXYZAngstWrtParticleCenter contains information on the icosahedrally refined offset of each subparticle in X,Y,Z with respect to the whole particle center. rlnCustomRelativePose contains the relative pose of the subparticle with respect to the icosahedrally refined orientation. rlnCustomRelativePose is stored in quaternion format rather than the standard RELION Euler angles, rlnAngleRot, rlnAngleTilt, rlnAnglePsi.Pentavalent and hexavalent capsomers were extracted in ISECC using the following commands, respectively:Correlational analysis was performed using the ISECC_local_motions script. Briefly, this parses the locally refined pentavalent and hexavalent capsomer star files to evaluate local deltas for subparticle origins and poses as compared to their idealized, icosahedrally derived, starting values. Coupled with the new metadata identifiers, rlnCustomVertexGroup and rlnCustomOriginXYZAngstWrtParticleCenter, this identifies deviation of each capsomer from idealized icosahedral symmetry on a per-particle basis.max corresponded to the particle radius as defined by the distance between the center of capsomer and the particle center. Despite a 5% tolerance being larger than necessary for a dataset of this size, it was chosen to allow for easy comparison to future datasets with a more modest particle number. This geometry minimizes contribution to distance along the Z-axis, which unlike X or Y cannot be locally refined. These capsomers are easy identified using the Z parameter within rlnCustomOriginXYZAngstWrtParticleCenter. The locally refined XY distance between polar opposite pentavalent capsomers was calculated for the 50,224 qualifying particles and compared to the Z-flattened icosahedrally-derived distance, producing a difference ratio. It is important to note that this analysis cannot capture the Z-component of any diameter deviation, necessitating the selection criteria described above.Deviation in particle diameter was calculated for all particles that satisfied selection criteria, namely, a pair of pentavalent capsomers within ± 5% (12.9 Å) of the central plane (Z = 0), where zCapsomer distance analysis was conducted on all particles with a hexavalent subparticle center within 25 Å x,y distance of the whole particle center. Capsomers with a potential doppelganger within 15 Å x,y distance on the opposite face of the capsid were excluded from this analysis to avoid the risk of subparticle identity swap during local refinement. Neighboring subparticles were then identified using the rlnCustomVertexGroup parameter, allowing the geometric relationships between capsomers to be distinguished (see the four relationships listed in Fig. Supplementary Information.