Fibril Formation Research Articles

Biophysical and spectroscopical insights into structural modulation of species in the aggregation pathway of superoxide dismutase 1

Superoxide dismutase 1 (SOD1) aggregation is implicated in the development of Amyotrophic Lateral Sclerosis (ALS). Despite knowledge of the role of SOD1 aggregation, the mechanistic understanding remains elusive. Our investigation aimed to unravel the complex steps involved in SOD1 aggregation associated with ALS. Therefore, we probed the aggregation using ThT fluorescence, size-exclusion chromatography, and surface-enhanced Raman spectroscopy (SERS). The removal of metal ions and disulfide bonds resulted in the dimers rapidly first converting to an extended monomers then coming together slowly to form non-native dimers. The rapid onset of oligomerization happens above critical non-native dimer concentration. Structural features of oligomer was obtained through SERS. The kinetic data supported a fragmentation-dominant mechanism for the fibril formation. Quercetin acts as inhibitor by delaying the formation of non-native dimer and soluble oligomers by decreasing the elongation rate. Thus, results provide significant insights into the critical steps in oligomer formation and their structure.

A study of alpha-synuclein and poly(N-isopropylacrylamide) complex formation through detailed atomistic simulations.

This work presents an investigation of the influence of poly(N-isopropylacrylamide) (PNIPAM) polymer on the structural dynamics of intrinsically disordered alpha-synuclein (α-syn) protein, exploring the formation and intricate features of the resulting α-syn/PNIPAM complexes. Using atomistic molecular dynamics (MD) simulations, our study analyzes the impact of initial configuration, polymer molecular weight, and protein mutations on the α-syn and the α-syn/PNIPAM complex. Atomistic simulations, of a few μs, of the protein/polymer complex reveal crucial insights into molecular interactions within the complex, emphasizing a delicate balance of forces governing its stability and structural evolution. Our findings indicate that PNIPAM polymer engages in significant non-polar interactions with the non-amyloid component (NAC) region of α-syn, which plays a crucial role in fibril formation, under various conditions such as the mutations in the protein structure and polymer chain length. Especially the PNIPAM polymer with a 40mer monomer exhibits a stabilizing effect on the structural properties of the protein, reducing intramolecular interactions that contribute to misfolding. These findings, which delve into protein/polymer interactions, hold promise as potential guidance for therapeutic strategies in various neurodegenerative disorders.

Lipid-induced condensate formation from the Alzheimer’s Aβ peptide triggers amyloid aggregation

The onset and development of Alzheimer's disease is linked to the accumulation of pathological aggregates formed from the normally monomeric amyloid-β peptide within the central nervous system. These Aβ aggregates are increasingly successfully targeted with clinical therapies at later stages of the disease, but the fundamental molecular steps in early stage disease that trigger the initial nucleation event leading to the conversion of monomeric Aβ peptide into pathological aggregates remain unknown. Here, we show that the Aβ peptide can form biomolecular condensates on lipid bilayers both in molecular assays and in living cells. Our results reveal that these Aβ condensates can significantly accelerate the primary nucleation step in the amyloid conversion cascade that leads to the formation of amyloid aggregates. We show that Aβ condensates contain phospholipids, are intrinsically heterogeneous, and are prone to undergo a liquid-to-solid transition leading to the formation of amyloid fibrils. These findings uncover the liquid-liquid phase separation behavior of the Aβ peptide and reveal a molecular step very early in the amyloid-β aggregation process.

Antarctic Krill Protein Amyloid Fibrils as a Novel Iron Carrier for the Improvement of Iron Deficiency.

Iron fortification with food supplements remains the primary dietary strategy for improving iron deficiency anemia (IDA). This study used Antarctic krill protein for fibrillar design to form an Antarctic krill protein amyloid fibril (AKAF). The results indicated that peptides generated by proteolysis were a prerequisite for fibril assembly, forming elongated fibril structures and cross-linking upon heating. During this process, hydrogen bonds were rearranged, forming ordered β-sheet conformations (49.36 ± 0.21%); π-π stacking interactions among aromatic residues contributed to fibril formation. Further studies showed that AKAF effectively maintained iron in a bioavailable state and exhibited a high binding capacity (60.67 ± 0.69%). Moreover, the AKAF-iron complex markedly ameliorated hematological abnormalities in IDA mice, enhanced iron storage in the liver and spleen, and positively influenced the expression of iron homeostasis genes. This complex was also effective in alleviating gastric inflammatory responses induced by IDA. Overall, AKAF holds promise as an efficient iron delivery carrier.

Unveiling an innovative sustainable blue resource-ecofriendly extraction technique towards a circular economy: Optimization of natural deep eutectic solvent and ultrasonication synergistic pathways for type-I collagen refined recovery from discarded fish scales.

A vast sum of fish waste is being annually discarded by marine fishing industries imposing serious environmental pollution concerns. However, these aquatic discarded matters are captivating sources of collagen, a fibrous protein with eminent social and economic relevance. Collagen is conventionally recovered using outdated complex processes requiring many reagents, multiple steps, and extended periods. Hereupon, the current project is the first work on the isolation of Seabass fish scales (FSC) type-I collagen, with preserved secondary and triple helical structures of the native collagen, developing a simple, green, cost-effective, and eco-friendly methodology, utilizing sustainable natural deep eutectic solvents (NADES)-assisted ultrasonication (US) technical route. The operational conditions were optimized based on the one-factor-at-a-time modeling to maximize the yield with no alteration of collagen integrity. Recorded data confirmed type-I collagen with preserved triple helix integrity and thermal stability, improved bio-functionalities, in vitro fibril formation, and functional performances. Finally, the in vitro hemolysis and cytotoxicity tests confirmed the extracted collagens biocompatibility, demonstrating the feasibility of Seabass FSC waste and a NADES-coupled US brief process (20 min) to establish a more sustainable eco-friendly pathway to isolate high-quality type I-collagen, as an attempt to rise industries awareness about wastes valorization within the scheme of circular economy.

Transient interactions between the fuzzy coat and the cross-β core of brain-derived Aβ42 filaments.

Several human disorders, including Alzheimer's disease (AD), are characterized by the aberrant formation of amyloid fibrils. In many cases, the amyloid core is flanked by disordered regions, known as fuzzy coat. The structural properties of fuzzy coats, and their interactions with their environments, however, have not been fully described to date. Here, we generate conformational ensembles of two brain-derived amyloid filaments of Aβ42, corresponding respectively to the familial and sporadic forms of AD. Our approach, called metadynamic electron microscopy metainference (MEMMI), provides a characterization of the transient interactions between the fuzzy coat and the cross-β core of the filaments. These calculations indicate that the familial AD filaments are less soluble than the sporadic AD filaments, and that the fuzzy coat contributes to solubilizing both types of filament. These results illustrate how the metainference approach can help analyze cryo-EM maps for the characterization of the properties of amyloid fibrils.

Lipidic folding pathway of α-Synuclein via a toxic oligomer

Aggregation intermediates play a pivotal role in the assembly of amyloid fibrils, which are central to the pathogenesis of neurodegenerative diseases. The structures of filamentous intermediates and mature fibrils are now efficiently determined by single-particle cryo-electron microscopy. By contrast, smaller pre-fibrillar α-Synuclein (αS) oligomers, crucial for initiating amyloidogenesis, remain largely uncharacterized. We report an atomic-resolution structural characterization of a toxic pre-fibrillar aggregation intermediate (I1) on pathway to the formation of lipidic fibrils, which incorporate lipid molecules on protofilament surfaces during fibril growth on membranes. Super-resolution microscopy reveals a tetrameric state, providing insights into the early oligomeric assembly. Time resolved nuclear magnetic resonance (NMR) measurements uncover a structural reorganization essential for the transition of I1 to mature lipidic L2 fibrils. The reorganization involves the transformation of anti-parallel β-strands during the pre-fibrillar I1 state into a β-arc characteristic of amyloid fibrils. This structural reconfiguration occurs in a conserved structural kernel shared by a vast number of αS-fibril polymorphs including extracted fibrils from Parkinson’s and Lewy Body Dementia patients. Consistent with reports of anti-parallel β-strands being a defining feature of toxic αS pre-fibrillar intermediates, I1 impacts viability of neuroblasts and disrupts cell membranes, resulting in an increased calcium influx. Our results integrate the occurrence of anti-parallel β-strands as salient features of toxic oligomers with their significant role in the amyloid fibril assembly pathway. These structural insights have implications for the development of therapies and biomarkers.

Chaperone-Mediated Heterotypic Phase Separation Prevents the Amyloid Formation of the Pathological Y145Stop Prion Protein Variant.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that arrests the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

Therapeutic Advantages of Nanoparticle-Impregnated Lysozyme Conjugates toward Amyloid-β Fibrillation and Antimicrobial Activity.

The interaction of protein with nanoparticles (NPs) of varying shape and/or size boosts our understanding on their bioreactivity and establishes a comprehensive database for use in medicine, diagnosis, and therapeutic applications. The present study explores the interaction between lysozyme (LYZ) and different NPs like graphene oxide (GO) and zinc oxide (ZnO) having various shapes (spherical, 's', and rod-shaped, 'r') and sizes, focusing on their binding dynamics and subsequent effects on both the protein fibrillation and antimicrobial properties. Typically, GO is considered a promising medium due to its apparent inhibition and prolonged lag phase for LYZ fibrillation. However, the present results showed that spherical ZnO NPs (sZnO) offer superior efficacy in modulating fibrillation with an extended lag time of about 158.70 h, further emphasizing the importance of detailed investigation on the nanomaterial characteristics and fibril formation kinetics beyond initial observations. The experimental findings further confirmed a strong correlation between the binding affinity of NPs to the native protein and their effective inhibition of protein denaturation, ultimately preventing fibril formation. Interestingly, the lysozyme nanoconjugates showed intriguing bactericidal effects, as confirmed through the agar plate assay and SEM imaging, over the native protein. Overall, this study shows that appropriate bionanomaterials can exhibit multifunctional properties, which paves the way for a deeper investigation of NP characteristics, ultimately benefiting a wide array of intriguing research.

Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis.

Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis

Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.

α-Synuclein interaction with POPC/POPS vesicles.

We have investigated the adsorption of the amyloid-forming protein α-Synuclein (αSyn) onto small unilamellar vesicles composed of a mixture of zwitterionic POPC and anionic POPS lipids. αSyn monomers adsorb onto the anionic lipid vesicles where they adopt an α-helical secondary structure. The degree of adsorption depends on the fraction of anionic lipid in the mixed lipid membrane, but one needs to consider the electrostatic shift of the serine pKa with increasing fraction of POPS. The vesicles with adsorbed αSyn monomers are kinetically stable. However, after fibrils have been formed, here triggered by the addition of a small concentration of pre-formed fibrils (seeds), we observed that the average vesicle size increased by approximately a factor of two. This increase in the vesicle size can be explained by vesicle fusion taking place during the fibril formation process.

Gut-Heart Axis: Microbiome Involvement in Restrictive Cardiomyopathies.

An intriguing aspect of restrictive cardiomyopathies (RCM) is the microbiome role in the natural history of the disease. These cardiomyopathies are often difficult to diagnose and so result in significant morbidity and mortality. The human microbiome, composed of billions of microorganisms, influences various physiological and pathological processes, including cardiovascular health. Studies have shown that gut dysbiosis, an imbalance in the composition of intestinal bacteria, can contribute to systemic inflammation, a key factor in many cardiovascular conditions. An increase in gut permeability, frequently caused by dysbiosis, allows bacterial endotoxins to enter the bloodstream, activating inflammatory pathways that exacerbate cardiac dysfunction. Recent reports highlight the potential role of microbiome in amyloidogenesis, as certain bacteria produce proteins that accelerate the formation of amyloid fibrils. Concurrently, advancements in amyloidosis treatments have sparked renewed hopes, marking a promising era for managing these kinds of diseases. These findings suggest that the gut-heart axis may be a potential factor in the development and progression of cardiovascular disease like RCM, opening new paths for therapeutic intervention. The aim of this review is to provide a detailed overview of the gut-heart axis, focusing on RCM.

Repurposing of Agrochemicals as ATTRv Amyloidosis Inhibitors.

Transthyretin (TTR), a plasma protein, undergoes transformation into amyloid fibers, leading to ATTRv amyloidosis, a disease characterized by organ deposition of TTR amyloid fibrils and subsequent organ failure. Developing compounds that bind and kinetically stabilize TTR is a crucial strategy in the treatment of ATTRv amyloidosis. In this study, we narrowed 651 pesticide-related compounds down to 14 possible TTR binders through in silico screening; subsequent in vitro analysis revealed that 7 of them exhibited amyloid fibril formation inhibition activity. The herbicide components bromoxynil (6) and ioxynil (21) showed especially high ligand efficiency and efficiently inhibited amyloid fibril formation of amyloidogenic V30M-TTR. Additionally, aclonifen (9) exhibited moderate fibril formation inhibition activity, but showed selective binding to TTR comparable to that of tafamidis. While improvement is needed to the selective TTR-binding or fibril formation inhibition activity, the compounds identified herein are promising lead candidates for the development of ATTRv amyloidosis therapeutics.

Characterization and modulation of human insulin degrading enzyme conformational dynamics to control enzyme activity.

Insulin degrading enzyme (IDE) is a dimeric 110 kDa M16A zinc metalloprotease that degrades amyloidogenic peptides diverse in shape and sequence, including insulin, amylin, and amyloid-β, to prevent toxic amyloid fibril formation. IDE has a hollow catalytic chamber formed by four homologous subdomains organized into two ~55 kDa N- and C- domains (IDE-N and IDE-C, respectively), in which peptides bind, unfold, and are repositioned for proteolysis. IDE is known to transition between a closed state, poised for catalysis, and an open state, able to release cleavage products and bind new substrate. Here, we present five cryoEM structures of the IDE dimer at 3.0-4.1 Å resolution, obtained in the presence of a sub-saturating concentration of insulin. Analysis of the heterogeneity within the particle populations comprising these structures combined with all-atom molecular dynamics (MD) simulations permitted a comprehensive characterization of IDE conformational dynamics. Our analysis identified the structural basis and key residues for these dynamics that were not revealed by IDE static structures. Notably arginine-668 serves as a molecular latch mediating the open-close transition and facilitates key protein motions through charge-swapping interactions at the IDE-N/C interface. Our size-exclusion chromatography-coupled small-angle X-ray scattering and enzymatic assays of an arginine-668 to alanine mutant indicate a profound alteration of conformational dynamics and catalytic activity. Taken together, this work highlights the power of integrating experimental and computational methodologies to understand protein dynamics, offers the molecular basis of unfoldase activity of IDE, and provides a new path forward towards the development of substrate-specific modulators of IDE activity.

Unveiling the inhibition mechanism of host-defense peptide cathelicidin LL-37 on the amyloid aggregation of the human islet amyloid polypeptide.

The aberrant aggregation of the human islet amyloid polypeptide (hIAPP) is a hallmark of type II diabetes. LL37, the only cathelicidin host-defense peptide in humans, plays essential roles in antimicrobial and immunomodulatory activities. Mounting evidence indicates that LL37 can inhibit the amyloid aggregation of hIAPP, suggesting possible interplays between infections and amyloid diseases while the mechanism remains unclear. In this paper, we explored the molecular interactions between hIAPP and LL37 using all-atom discrete molecular dynamics (DMD), a novel and predictive molecular dynamics engine with improved sampling efficiencies. We found that the LL37 peptides can effectively interact with hIAPP in monomer, oligomer, and fibril states driven by hydrophobic associations and pi-pi interactions. Specifically, the hydrophobic residues in the N- and C-termini of LL37 peptides can firmly bind with the monomeric and oligomeric hIAPP, especially in the amyloidogenic regions, to prevent the self-interactions of amyloidogenic regions and thus hinder the formation of amyloid fibrils. Furthermore, LL37 can bind to the elongation surfaces of the hIAPP fibril seeds with geometric incompatibility for monomer addition to block the fibril growth. Together, we identified the crucial residues and key driving forces for the interactions between LL37 and hIAPP peptides and revealed the related dynamics and conformational changes. The uncovered mechanism can contribute to a better understanding of the pathological links between microbial infections and amyloid diseases and guide the designs of novel therapies combining antimicrobial and anti-amyloid functions.

Different types of anions mediated the formation of rice glutelin fibrils: Aggregation behaviors and structural characteristics.

Anions have more pronounced effect on the aggregation power of proteins than cations. Herein, the effect of different types of anions on rice glutelin (RG) based fibrils formation was investigated. The fibrils yield and growth rate of RG were enhanced with various anions, due to the specific ions effect and intermolecular interaction. It was observed that the fibrillization rate of RG followed the specific order: Br->I->F->NO3->Cl->CH3COO->ClO-. In addition, the chaotropes (e.g., I-) interacted more favorably with hydrophobic residues to form needle-like periodic structure, whereas the kosmotropes (e.g., CH3COO-) preferred to form larger fibrils clusters due to the stronger shielding effect. Consequently, various anions profoundly influenced the unfolding, dimerization, reorganization of protein, as well as the formation of crucial β-sheet structure. This study helps to understand how the structure of fibrils can be tuned through different anions.

In Situ Polymerization and Synthesis of UHMWPE/Carbon Fiber Composites.

Carbon-fiber-reinforced composites of ultra-high-molecular-weight polyethylene (UHMWPE) are not easily prepared because of their high viscosity, although they can be advantageous in advanced engineering applications due to their superior mechanical properties in combination with their low specific weight and versatility. Short polyacrylonitrile-based carbon-fiber-reinforced UHMWPE composites with fiber contents of 5, 10, and 15 wt.% could easily be prepared using in situ ethylene polymerization. Therefore, MgCl2 was generated at the Brønsted acidic groups of the fiber surface by employing a reaction between the co-catalysts Mg(C4H9)2 and AlEt2Cl. Titanation with TiCl4 resulted in a catalyst directly on the fiber surface. The catalyst polymerized ethylene (2 bar pressure at 50 °C), forming a UHMWPE matrix near the surface; its fragmentation led to polymer particles associated with the fiber. The catalyst activity on the fiber surface of untreated (CF-Pr, 3.48 ± 0.24 wt.%) and oxidized fibers (CF-Ox, 7.41 ± 0.03 wt.%) was 20% lower. CF-Pr compression-molded samples showed tensile strengths of up to 50.4 ± 1.3 MPa, while CF-Ox samples reached 39.1 ± 0.6 MPa, surpassing the performance of composites prepared by melt compounding. The stiffness of 1.58 ± 0.17 GPa for a melt-compounded material was lower than the 3.24 ± 0.10 GPa for CF-Pr and 2.19 ± 0.07 GPa for CF-Ox composites. A fracture examination showed fiber pull-outs, matrix residues on the fibers, and the formation of some extensional polymer fibrils. The latter explains the higher stress at yield and the breakage of the CF-Pr based composites in particular. The potential of in situ polymerized UHMWPE composites for the utilization in high-performance structural applications is thus demonstrated.

Ultrasound-assisted heat treatment: Accelerating rice glutelin fibrils formation and enhancing emulsifying properties.

The self-assembly of rice glutelin (RG) into RG fibrils (RGFs) represents a promising strategy for enhancing its functional properties. In this study, we investigated the effects of ultrasonic pretreatment on the fibrillation kinetics, structural characteristics, and functional properties of RGFs. The results indicated that ultrasonic pretreatment facilitated the unfolding of RG, resulting in an increased H0 and β-sheet, thereby accelerating the formation of RGFs and enhancing the fibril conversion rate. Thioflavin T fluorescence spectroscopic analysis confirmed the formation of numerous cross β-sheet structures following 4 h of heating; however, ultrasonic pretreatment reduced this duration to just 2 h. Additionally, the ζ-potential and solubility of RGFs were significantly improved following ultrasonic pretreatment. TEM revealed that the URG-6 fibril sample exhibited the smallest diameter (3.81-5.27 nm) and greatest length (1109.92-1946.21 nm), demonstrating a high aspect ratio. Furthermore, ultrasonic pretreatment enhanced the emulsifying properties of RGFs, with the URG-6 emulsion achieving the highest EAI (147.61 m2/g) and ESI (134.67 min), along with the smallest droplet sizes. This study provides the basis for the development of RGFs and broadens their application in the food industry.

Tau destabilization in a familial deletion mutant K280 accelerates its fibrillization and enhances the seeding effect.

Tauopathies cover a range of neurodegenerative diseases in which natively unfolded tau protein aggregates and spreads in the brain during disease progression. To gain insights into the mechanism of tau structure and spreading, here, we examined the biochemical and cellular properties of human full-length wild-type and familial mutant tau, ΔK280, with a deletion at lysine 280. Our results showed that both wild-type and mutant tau are predominantly monomeric by analytical ultracentrifugation. The mutant tau may lose intramolecular contacts and is significantly destabilized assessed by cross-linking mass spectrometry and urea denaturation. Moreover, the mutant tau displayed accelerated fibril formation compared to the wild-type tau. Upon cross-seeding, the wild-type tau was seeded more easily by wild-type seeds than mutant seeds showing that homotypic seeding is more efficient. The wild-type tau was successfully converted to fibrils with mutant signatures by mutant seeds. Live cell cross-correlation fluorescence spectroscopy studies indicated that wild-type tau forms trimeric species and the mutant tau forms a larger assembly and processes higher cell-to-cell transmission. Overall, these findings shed light on the fundamental mechanisms of tau structure/stability, aggregation, and seeding to facilitate future therapeutic development for tauopathies.