self - assembly of proteins 
 into long fibrillar aggregates with similar 
 cross- sheet structures is the molecular signature of amyloid 
 diseases , including alzheimer s disease , tauopathies , prion 
 diseases , type ii diabetes , and rheumatoid arthritis . while the molecular mechanisms underlying amyloid pathogenesis 
 remain intensely debated , the toxicity of globular oligomeric intermediates 
 of the fibril assembly process is a prominent suspect . yet , amyloid oligomers are considered short - lived intermediates 
 emerging during amyloid assembly that require extrinsic stabilization 
 for studying their properties . furthermore , 
 these oligomeric intermediates might be off - pathway from mature fibril 
 formation and metastable against seeding with mature fibrils . this raises the question how short - lived , off - pathway intermediates 
 of the assembly process can initiate the various pathologies associated 
 with amyloid diseases and promote their spread throughout tissues . however , our understanding of the properties of amyloid intermediates 
 and their ability to promote amyloid formation of monomers are typically 
 derived from in vitro fibril growth assays often 
 confined to a narrow range of partially denaturing solution conditions . 
 comparatively little is known about the behavior of distinct amyloid 
 species close to physiological solution conditions , in particular 
 whether they remain stable instead of dissolving or precipitating 
 and whether they are capable of seeding growth from native monomers . multiple experimental observations and theoretical considerations have suggested 
 that changes in solution environment ( ph , temperature , ionic composition , 
 and protein concentration ) could alter basic properties of amyloid 
 aggregates , including their assembly mechanisms , their aggregation 
 propensities , and their interactions with monomers . investigating 
 the aggregation propensity of different amyloid templates with native 
 monomers also addresses recent suggestions that amyloid oligomers 
 might assume prion - like characteristics that contribute to their spread 
 in neurodegenerative diseases . we set 
 out to investigate whether amyloid oligomers , protofibrils , and mature 
 fibrils remained intact at physiological temperatures , and whether 
 any of them could induce amyloid formation from lysozyme in its native 
 fold . for these experiments we used hen egg - white lysozyme and its 
 well - defined oligomeric and fibrillar assembly pathways as our model 
 system . hen egg - white lysozyme 
 ( hew - l ) is a small enzyme of 14.3 kda molecular 
 weight with well - defined biochemical and physical properties . point 
 mutations in human lysozyme underlie non - neuropathic systemic forms 
 of human amyloidosis , with dangerous disruptions to kidney and liver 
 function . hew - l undergoes a well - defined 
 cooperative unfolding transition as a function of temperature , thereby 
 permitting us to determine how different amyloid species interact 
 with natively folded vs partially unfolded monomers . thermal denaturation 
 also induces spontaneous amyloid formation of lysozyme in 
 vitro.(19,20 ) fibril formation of lysozyme 
 switches sharply from generating rigid filaments at low ionic strength 
 to producing compact oligomeric intermediates at higher ionic strength , 
 which subsequently nucleate curvilinear protofibrils . the existence of an oligomeric vs fibrillar assembly pathways 
 has been reported for multiple amyloid proteins including amyloid- , 
 beta-2 microglobulin , and polyglutamine . for some of these systems , the oligomers and/or protofibrils are 
  on pathway  to mature fibril formation , while the globular oligomer and protofibrils of lysozyme and beta-2 
 microglobulin remain off - pathway . furthermore , lysozyme oligomers , 
 protofibrils , and rigid filaments all develop -sheet peaks 
 in their ir spectra that are considered diagnostic of amyloid structures . differences in their ir peak wavenumber and 
 morphology imply that lysozyme oligomers and protofibrils are amyloidolytic 
 structures that are distinct from their rigid filament counterparts . the investigation into potential prion - like 
 activity of lysozyme 
 aggregates was motivated by observations that oligomer or protofibril 
 growth at partially denaturing temperatures appeared to continue , 
 albeit at a very slow pace , long after solutions had been returned 
 to room temperature . we therefore tested whether oligomers , protofibrils , 
 and mature fibrils remained intact and soluble upon return to physiological 
 temperatures and ph , and whether they developed prion - like characteristics 
 of self - replication from native substrate under these conditions . while we intended to confine these experiments to the physiological 
 ph of 7 , we found that thermal denaturation of hew - l at ph 7 readily 
 produced oligomers and protofibrils but no filaments . including ph 
 3 in the experiments allowed us to generate all three aggregate species 
 by simply changing the nacl concentration . equally important , ph 3 preserves the native fold at 37 c 
 and the cooperative unfolding of lysozyme s secondary and tertiary 
 structure near 70 c . at more acidic ph values , this cooperativity 
 breaks down leading to partial loss of tertiary structure prior to 
 secondary structure unfolding . in addition , 
 ph 3 and 37 c avoids the noticeable monomer hydrolysis at more 
 acidic ph values . we also performed cross - seeding 
 experiments by transferring filaments and oligomers / protofibrils generated 
 at ph 3 as seeds into fresh monomer solutions at ph 7 . to assess aggregate 
 stability , aggregation kinetics , and aggregate structures during seeding 
 experiments , we relied on a broad range of complementary experimental 
 techniques including tht fluorescence , static and dynamic light scattering 
 ( sls , dls ) , atomic force microscopy ( afm ) , and attenuated total reflectance 
 fourier transform infrared spectroscopy ( atr - ftir ) . two times 
 recrystallized , dialyzed , 
 and lyophilized hen egg white lysozyme ( hew - l ) was purchased from 
 worthington biochemicals ( lakewood nj ) and used for all experiments . all other chemicals were purchased from fisher scientific ( pittsburgh , 
 pa ) and were reagent grade or better . typically 
 hew - l was dissolved in 20 mm hepes at ph 7 or 10 mm of sodium citrate 
 at ph 3 and filtered consecutively through 220 and 50 nm syringe filters . lysozyme concentrations were determined from 
 uv absorption using a280 = 2.64 ml mg cm . for oligomer / protofibril 
 formation at ph 7 , solutions were incubated with either 40 or 80 mm 
 nacl at 70 c for 1.56 h. at ph 3 , oligomer / protofibril 
 formation was induced by incubation lysozyme with 250 mm nacl at 70 
 c for 410 h. for amyloid filament growth , solutions 
 were incubated at ph 3 with 75100 mm nacl for 2030 
 h. seeds were separated from lysozyme monomers using 50 kda mw centricon 
 filters , spun at 1400 g , and rinsed multiple times 
 with the appropriate growth buffer . 100150 l of isolated 
 seeds in buffer were collected from the top reservoir of the filter , 
 and the seed concentration was determined from uv absorption . for 
 seed growth experiments , native lysozyme solutions were mixed with 
 520% ( v / v ) of amyloid seed solutions and incubated for 514 
 days . for seed stability experiments , oligomers / protofibrils were 
 added at 1020% ( v / v ) to buffer and incubated up to 50 h , and 
 up to 100 h for filaments . for experiment on monomeric dependence 
 of seeded oligomer / protofibril growth , 10% of isolated seeds were 
 incubated with 0.220 mg / ml of monomeric lysozyme solution 
 at 37 c and ph 7 . for cross - seeding experiments , protofibrils 
 and filaments generated at ph 3 were separated from monomers and brought 
 to ph 3 via dialysis against ph 7 buffer . amyloid 
 filaments also tended to precipitate but , after passing through 0.8 
 m syringe filters , resolubilized . these fibrils were concentrated 
 to 20 mg / ml right before seeding with fresh monomers at ph 7 . static and dynamic 
 light scattering ( sls and dls ) measurements were performed using a 
 zetasizer nano s ( malvern instruments , worchestershire , uk ) . for in situ measurements , autocorrelation functions of scattered 
 light were collected using acquisition times of 3 min and converted 
 into particle - size distributions using the  narrow modes  
 or general purpose  algorithms provided with the zetasizer 
 nano s. changes in scattering intensity were derived from the count 
 rates of the avalanche photodiode photon detector . amyloid aggregates were imaged 
 by depositing samples on freshly cleaved mica and drying them with 
 nitrogen . images were acquired with an mfp-3d atomic - force microscope 
 ( asylum research , santa barbara , ca ) using nsc36/noal ( mikromasch , 
 san jose , ca ) silicon tips with nominal tip radii of 10 nm . the cantilever 
 was driven at 6070 khz in alternating current mode . images 
 at 512  512 pixel resolution were acquired at a scan rate of 
 0.5 hz . tht fluorescence 
 measurements were performed on a fluoromax-4 spectrofluorometer ( horiba 
 jobin yvon , edison , nj ) using excitation and emission wavelengths 
 of 445 and 485 nm , respectively . for tht measurements , small aliquots 
 of the hew - l solutions were removed and mixed 100:1 with a tht stock 
 solution , resulting in a final tht concentration of 10 m . attenuated total reflectance fourier - transform infrared spectroscopy 
 ( atr- ftir ) was performed on a bruker optik vertex 70 ( ettlingen , 
 germany ) spectrometer with a mid - infrared source and a pyroelectric 
 dlatgs ( deuterated l - alanine doped triglycene sulfate ) room 
 temperature detector . 30 l of protein solution were placed 
 on the thermostated silicon crystal of a bioatrcell ii ( harrick scientific 
 products , inc . , 400800 scans 
 at 1 cm step size were recorded and 10 such runs 
 were averaged prior to data processing . first , we determined how lowering 
 the solution temperature from above the onset for monomer denaturation 
 ( 70 c ) to physiological values ( 37 c ) affected the stability 
 of the various amyloid species generated at either ph 3 or ph 7 . persistent 
 solubility and lack of dissociation of aggregates near physiological 
 temperatures is a prerequisite for their ability to propagate within 
 tissues and seed new amyloid formation there . we used transient thermal 
 denaturation , required to induce amyloid formation , and brought these preformed amyloid aggregates back to physiological 
 temperature . filaments or oligomers and protofibrils at ph 3 were 
 formed by incubating 1.4 mm lysozyme at 70 c with either 100 
 mm or 250 mm nacl , respectively . at ph 7 only amyloid oligomer and 
 protofibril growth was observed . oligomers at ph 7 were generated 
 by incubating 1.4 mm lysozyme with 40 mm nacl at 70 c for 1.5 
 h or less . after 2 h protofibril nucleation was detected by dls , with 
 the proportion of protofibrils in solution increasing with incubation 
 period . in all cases amyloid growth was monitored in situ using static and dynamic light scattering ( dls ) . afm images of amyloid 
 aggregates generated at either ph 3 or ph 7 showed the characteristic 
 morphologies associated with either amyloid filaments ( figure 1a ) or oligomers and protofibrils ( figures 1b and 6a ) . they displayed the same kinetic , tinctorial , and structural 
 features as those previously described at ph 2 and 7 . afm images of ( a ) lysozyme filaments and 
 ( b ) protofibrils formed during incubation at 70 c and ph 3 in 
 100 or 250 mm nacl , respectively . . images of oligomers / protofibrils at ph 7 are 
 provided in figure 6a . static light scattering 
 ( iscat ) indicates that at 37 c ( c ) 
 filaments formed at ph 3 remain stable while ( d ) protofibrils decay 
 very slowly . inserts : particle size distributions obtained with dls at the beginning 
 ( --- ) and end ( ) of the incubation period . to evaluate their thermal stability in the absence 
 of monomers , seeds were separated from their monomer background using a 50 kda 
 recovery filter and subsequent resuspension in buffer . successful 
 separation and integrity of seeds was confirmed using dls and afm 
 imaging on the separated aggregates . the stability of isolated seeds 
 at 37 c was determined by monitoring their scattering intensity 
 and particle size distributions for a minimum of 24 h. amyloid filaments , 
 which represent the aggregate morphology most commonly associated 
 with amyloid diseases , remained stable at 37 c ( figure 1c ) . oligomer and protofibrils at ph 3 dissociated 
 slowly with a small monomer peak emerging in dls after about 10 h 
 of incubation ( figure 1 d ) . protofibrils 
 ( figure 1e ) and oligomers generated at ph 7 
 showed no signs of decay or self - assembly at 37 c . this stability 
 contrasts with the rapid dissociation of both isolated fibrils or 
 protofibrils at ph 3 and the rapid self - association of protofibrils 
 at ph 7 upon heating near 70 c ( data not shown ) . the gain in 
 thermal stability was not associated with any obvious changes to aggregate 
 sizes ( dls ) , morphologies ( afm ) , or structures ( atr - ftir ) . oligomers 
 and protofibrils are considered short - lived intermediates 
 of the assembly process . for lysozyme and beta-2 microglobulin , they 
 form along assembly pathways distinct from that for amyloid filament 
 assembly and are metastable against filament growth via seeding . however , upon lowering solution temperature to physiological values , 
 oligomers and protofibrils retained their solubility and displayed 
 either very low rates ( ph 3 ) or no discernible signs ( ph 7 ) of either 
 fragmentation or dissociation into monomers . the observed thermal 
 stability of filaments should not be considered self - evident either . this was highlighted by recent experiments on tau filaments which 
 readily formed at high temperatures , but dissolved when returned to 
 physiological temperatures . isolated 
 amyloid aggregates of lysozyme formed along either pathway , at a minimum , 
 dramatically reduce their rates of dissociation , fragmentation , and 
 self - association when lowering temperatures back into the physiological 
 range . the stability of various amyloid species close to physiological 
 conditions is relevant to the question how supposedly short - lived 
 and/or metastable amyloid species can migrate within tissues or even 
 the entire body , thereby spreading amyloid formation . at denaturing temperatures used 
 for seed growth , oligomers , protofibrils , and filaments all readily 
 grow from partially unfolded lysozyme monomers . at physiological temperatures spontaneous amyloid formation 
 from native proteins is not observed ( see also figure 4a below ) . we therefore tested whether the 
 above long - lived amyloid species could induce fresh amyloid growth 
 when added to native lysozyme maintained at physiological temperatures 
 and ph . initially we tested the behavior at ph 3 since it permitted 
 inclusion of rigid filaments . amyloid seeds of either type were diluted 
 10-fold into solutions of native folded lysozyme at ph 3 using the 
 same solution composition as during seed formation , but maintaining 
 the temperature at 37 c throughout . the particle size distributions 
 for fresh monomers seeded with amyloid filaments or mixtures of amyloid 
 oligomers and protofibrils are shown in figure 2a and d , respectively . amyloid filaments at various stages of assembly 
 were seeded into fresh monomers , but showed no discernible signs of 
 growth at 37 c over 3 days ( figure 2b , 
 c ) . this indicates that monomeric filaments can not utilize natively 
 folded lysozyme at ph 3/37 c as the substrate for continued 
 growth . in stark contrast , oligomer / protofibrils at ph 3 readily induced 
 aggregation of native lysozyme solutions . scattering intensity doubled 
 in as little as 18 h ( figure 2f ) , and the relative 
 size of the protofibril peak grew at the expense of the monomer peak 
 ( figure 2e ) . it is worth recalling that both 
 amyloid oligomers / protofibrils and filaments at ph 3 assemble spontaneously , 
 and continue to grow , once lysozyme is partially unfolded ( t = 70 c ) . hence , both oligomers / protofibrils and 
 filaments form suitable templates for amyloid growth from partially 
 unfolded monomers . upon return to 37 c the ability of oligomers 
 and protofibrils to seed fresh growth at 37 c , combined with 
 the inability of filaments to do so , implies the absence of even small 
 percentages of partially unfolded solution states which would have 
 supported continued growth of either template . lysozyme oligomers 
 and protofibrils , but not filaments , induce 
 growth of native lysozyme . . the short and 
 long monomeric filament peaks ( s - mf , l - mf ) are characteristic for 
 this assembly pathway . filament - seeded 
 monomer solutions displayed no changes in ( b ) size distribution or 
 ( c ) scattering intensity during 3 days of incubation . ( d ) corresponding 
 size distribution for mixtures of oligomers ( merged with monomer peak ) 
 and protofibrils ( 40 nm peak ) seeded into monomers . within 16 h ( f ) 
 the scattering intensity from oligomers / protofibril - seeded samples 
 more than doubled . at the same time ( e ) the protofibril peak p grew 
 at the expense of the monomer peak m. to ensure that monomers retain a native fold , we repeated 
 the seeding 
 experiments near the physiological ph of 7 . after generating either 
 oligomers or mixtures of oligomers and protofibril at 70 c at 
 various stages of self - assembly , they were diluted 5- to 20-fold into 
 1.4 mm of fresh lysozyme at 37 c . seeded solutions were incubated 
 for a period of time ranging from 4 days to 2 weeks . solutions of 
 native lysozyme at 37 c seeded with oligomers only ( figure 3a ) displayed kinetic growth signatures similar to 
 those for barrier - free oligomer formation at denaturing temperatures , 
 albeit at much reduced growth rates . this included 
 a prominent nucleation event associated with protofibril nucleation 
 ( figure 3b ) and an accelerating increase in 
 scattering intensity , particularly after protofibril nucleation , resulting 
 in a 7-fold scattering increase over 2 weeks ( figure 3c ) . seeding monomers with mixtures of protofibrils and oligomers 
 ( figure 3d ) resulted in noticeably faster increases 
 in protofibril radii ( figure 3e ) and scattering 
 intensities ( figure 3f ) . seeding native 
 lysozyme with oligomers and protofibrils at 
 ph 7 . particle size distribution derived from dls for solutions 
 of lysozyme monomers at ph 7 seeded with 20% ( v / v ) of ( a ) oligomers 
 or ( d ) mixtures of oligomers and protofibrils . labels indicate the 
 ( merged ) monomer / oligomer peak ( m / o ) and protofibril peak ( p ) . temporal 
 evolution in the peak radii for samples seeded with ( b ) oligomer - seeded 
 or ( c ) oligomer and protofibril . corresponding changes in scattering 
 intensity are displayed in panels ( c ) and ( f ) . the sample seeded with 
 oligomers nucleated protofibrils around 6 days ( see panel b ) . we performed a series of controls 
 to confirm that seeded growth 
 indeed represented induced aggregation of native monomers and required 
 amyloid oligomers / protofibrils instead of partially denatured monomers . these control experiments were performed at ph 7 and 37 c , because 
 it represents the biologically most relevant situation . incubating 
 native lysozyme monomers without seeds , as expected , yielded no signs 
 of aggregation for up to 3 months ( figure 4a ) . isolated oligomers 
 or protofibrils in the absence of lysozyme monomers also did not display 
 any tendency toward self - assembly at 37 c ( figure 1e ) . we also seeded fresh monomers with either transient denatured 
 lysozyme monomers or amyloid protofibrils , separated from each other 
 after seed growth at 70 c and brought to identical concentrations . only amyloid protofibrils induced aggregation of native lysozyme at 
 37 c , indicating that the observed aggregation indeed required 
 both amyloid seeds and native monomers . moreover , we determined whether 
 seeded aggregation did indeed consume fresh lysozyme monomers , instead 
 of reporting on some monomer - induced self - assembly process of seeds 
 only . toward that end concentrations of free monomers at various 
 times after seeding were determined by pelleting aggregates from aliquots 
 of growth solution via ultracentrifugation and measuring the residual 
 monomer concentration in the supernatant . seed - induced aggregation 
 depleted monomers to levels far exceeding the original seed concentration 
 ( 10% v / v ) . monomer depletion also correlated with aggregation - induced 
 increases in scattering intensity and particle sizes measured just 
 prior to ultracentrifugation ( figure 4c ) . seeded growth requires both oligomers / protofibrils and native 
 lysozyme , depletes native monomers . ( a ) lack of changes in 
 the hydrodynamic radius rh (  ) 
 or light scattering intensity iscat (  ) 
 for native lysozyme incubated at ph 7 and 37 c for one month . ( b ) changes in light scattering for lysozyme monomers incubated with 
 20% of either isolated protofibril (  ) or monomers (  ) , 
 separated from each other prior to seeding into fresh monomers . ( c ) 
 depletion of fresh lysozyme ( 20 mg / ml ) during seeded growth at 37 
 c with 15% ( v / v ) early stage purified protofibrils . residual 
 monomer concentrations in the supernatant (  ) after centrifugation 
 ( 2 h at 98  000 g ) , and light scattering intensities 
 (  ) from aliquots of seeded solution were monitored for approximately 
 4.5 days . the above data ( figures 2 and 3 ) establish that amyloid 
 oligomers and/or protofibrils , but 
 not filaments , were able to induce aggregation of native lysozyme 
 at physiological temperature . the capability of lysozyme oligomers 
 and protofibrils to seed fresh growth was preserved even at physiological 
 ph . the observed evolution in particle size distributions ( dls ) and 
 scattering intensities ( sls ) of seeded growth at physiological temperatures 
 replicated many of the distinct kinetics features ( temporal evolution 
 of the particle size distributions ; lag phase prior to protofibril 
 nucleation ; slow near - linear increase in mean protofibril radius ; 
 exponential growth in scattering intensity ) associated with lysozyme 
 fibril growth along the oligomeric pathway at denaturing temperatures . we attempted 
 to cross - seed protofibrils and filaments , generated during transient 
 denaturation at ph 3 , into solutions of native lysozyme at ph 7 . if successful , they also permit testing whether the 
 capacity for growth from native protein persists with changing solution 
 ph . protofibrils and filaments at ph 3 were generated as described 
 above and then either directly transferred to ph 7 or dialyzed to 
 ph 7 and separated from monomers . either way , oligomers and protofibrils 
 at any stage of assembly at ph 3 irreversibly precipitated from solution 
 at ph 7 making them unsuitable for seeding experiments . filaments 
 also showed signs of precipitation following dialysis , but could be 
 resolubilized after passing them through 0.8 m pore syringe 
 filters . filaments , resolubilized at ph 7 , were concentrated to 20 
 mg / ml and then seeded into fresh lysozyme at ph 7 and 37 c . 
 judged by the lack of change in scattering intensity or tht response , 
 filaments in the presence of monomers did not grow ( figure 5 ) . in contrast , protofibrils generated at ph 7 and 
 seeded into fresh monomers at the same concentration as filaments 
 displayed robust growth . seeds of monomeric 
 filaments were generated at ph 3 , separated from monomers , dialyzed , 
 and resolubilized at ph 7 . after concentrating seeds to 20 mg / ml , 
 they were diluted 10-fold into either 20 mg / ml of native lysozyme 
 or buffer only ( ph 7 , 40 mm nacl ) and incubated at 37 c . monomeric 
 filaments ( mf ) did not induce growth of hew - l monomers as indicated 
 by scattering intensity ( a ) or tht fluorescence emission ( b ) . in contrast , 
 protofibrils ( pf ) generated at ph 7 , concentrated ( 20 mg / ml ) , and 
 diluted 10-fold into fresh monomers readily induced their self - replication 
 ( a ) . protofibrils generated at ph 3 could not be used as seeds since 
 they precipitated irreversibly following transfer to ph 7 . to corroborate the amyloidogenic 
 character of aggregates formed at physiological temperature and ph 
 via seeded growth of native lysozyme we compared the aggregates 
 morphological , tinctorial , and structural characteristics to those 
 for protofibrils grown at denaturing temperatures . figure 6a displays afm images of lysozyme oligomers used for seeding and 
 the morphologies of the resulting aggregates formed after incubation 
 with native lysozyme at 37 c for 5 days and 3 weeks , respectively . following incubation , increasing numbers of highly flexible protofibrils 
 appeared that tended to fold onto themselves upon deposition onto 
 mica surfaces . aggregation in seeded lysozyme solutions at 37 c 
 also increased thioflavin t fluorescence , a well - established amyloid 
 indicator dye . for a given sample , fractional increases in light scattering 
 vs tht fluorescence intensity during incubation were of comparable 
 magnitude ( figure 6b ) , replicating features 
 of oligomer / protofibril growth at denaturing solution temperatures . ftir spectra for aggregates formed at either 
 37 or 70 c , after subtraction from those of native monomers , 
 highlight aggregation - induced structural changes within the amide - i 
 band ( figure 6c ) . aggregates emerging at the 
 late stages of incubation at 70 c or seeded growth at 37 c 
 displayed decreases in their percentage of native -helical 
 content ( 1660 cm ) that correlated with 
 their concurrent increases in -sheet structure between 1630 
 and 1610 cm . this latter band of wavenumbers is 
 considered diagnostic for -sheet formation associated with 
 amyloid fibril growth . we also find a 
 much weaker peak emerging near 1695 cm , which 
 has been associated with antiparallel -sheets in a amyloid 
 oligomers . the above kinetic , morphological , 
 tinctorial , and structural characteristics confirm that aggregates 
 formed at physiological conditions via seeding native lysozyme with 
 oligomers / protofibrils are indistinguishable from oligomers and protofibrils 
 grown from partially denatured lysozyme . therefore , lysozyme amyloid oligomers or protofibrils , when stabilized 
 at 37 c , do indeed induce their self - replication from native 
 lysozyme as the growth substrate . ( a ) afm images ( from left 
 to right ) of oligomeric seeds , and resulting 
 early stage protofibrils ( 5 days incubation ) and late stage protofibrils 
 ( 3 weeks of incubation ) grown via seeding of native lysozyme at ph 
 7 and 37 c . ( scale bar : 200 nm ; color scale : height in nm ) . ( b ) fractional increases in off - line tht fluorescence emission (  ) 
 and light scattering intensity (  ) during seeded growth of 
 native lysozyme . ( c ) normalized ftir difference spectra within the 
 amide i band for late - stage amyloid aggregates at ph 7 formed after 
 incubation at either 70 or 37 c , following subtraction of the 
 spectrum for lysozyme monomers . there are three major models widely considered 
 for amyloid self - assembly : templated assembly ( ta ) , nucleated polymerization ( np ) with variants accounting for secondary generation of new growth 
 ends via fragmentation of existing seeds ( np - f ) or via secondary surface nucleation of new seeds ( np - sn ) , and nucleated conformational conversion ( ncc ) . these models make distinct predictions for the dependence of growth 
 rates on both seed and monomer concentrations . specifically , ta growth 
 rates are reaction - limited by the number of seeds added to the system . nucleated polymerization and nucleated conformation conversion , in 
 turn , share characteristics of phase transitions . the 
 mechanisms of secondary generation incorporated into variants of nucleated 
 polymerization both predict exponential growth rates due to the multiplicative 
 increase of growth sites . however , fragmentation rates should be independent 
 of monomer concentration while secondary surface nucleation rates 
 will depend on both seed and monomer concentration . nucleated models 
 also presume behavior reminiscent of phase transitions , which requires 
 monomer concentrations beyond some finite solubility limit , i.e. supersaturation . to gain insights into what model might account for our observations 
 of oligomer / protofibril growth under native solution conditions , we 
 explored its dependence on seed age and monomer concentration . a fixed 
 concentration of lysozyme monomers was seeded with oligomers only 
 ( o ) , early stage protofibrils ( esp ) , or late - stage protofibrils ( lsp ) . figure 6a shows a semilog plot of the increases 
 in scattering intensity with incubation period , and linear fits through 
 these data . such an exponential increase implies 
 the action of a mechanism for self - replicating growth units from those 
 already present ( dn  n(t ) ) , similar to fragmentation or secondary 
 nucleation . the sample seeded with oligomers 
 only also displayed a dramatic increase in the exponential growth 
 rate near 5 days which coincided with the nucleation of protofibrils 
 reported by dls . therefore , upon protofibril nucleation , this self - replication 
 mechanism becomes more efficient or an additional mechanism is activated . the increases in growth rate with seed maturity suggest that the efficacy 
 of this mechanism might also increase with seed age , potentially through 
 a dependence on their contour length . to explore the mechanisms 
 of self - replication further , we tested 
 the dependence of growth on monomer concentration while keeping seed 
 concentration and age fixed . the initial slope of the temporal changes 
 in scattering intensity ( see figure 7a ) was 
 taken as a measure of the growth rate of the seeded samples . figure 7b is a log  log plot of the derived growth 
 rates vs monomer concentrations for samples seeded simultaneously 
 with 10% ( v / v ) of protofibrils from the same seed stock . the observed 
 growth rate increased in nearly direct proportion ( power law exponent 
 0.85  0.15 ) over 2 orders of magnitude in monomer concentration . it displays no signs of a zero - point offset , indicative of a finite 
 solubility associated with a phase transition . it also lacks signs 
 of slowing down at high monomer concentrations as expected for a reaction - limited 
 growth process . ( a ) natural logarithm of the scattering intensity 
 ( iscat ) for 20% ( v / v ) of amyloid oligomers 
 ( o ) , early stage 
 protofibrils ( esp ) , or late - stage protofibrils ( lsp ) incubated with 
 native lysozyme at 37 c and ph 7 . the break in the 
 growth rate for ( o ) coincided with the nucleation of protofibrils . ( b ) log  log plot of the aggregate growth rate for solutions 
 of 10% ( v / v ) of purified protofibrils seeded into increasing concentrations 
 of lysozyme monomers ( 0.220 mg / ml ) . aggregation rates were 
 equated to the slope of the factional increases in scattering intensity 
 near the onset of growth . a power law fit through the data ( --- ) yielded 
 an exponent of 0.85  0.16 . clearly , ta can not account for the exponential increase in 
 growth 
 rates seen in figure 7a . furthermore , the rate - limiting 
 step in ta is considered to be the conversion of monomers to the template 
 structure . this would imply saturating growth rates at elevated monomer 
 concentrations , which was not observed ( figure 7b ) . the strong dependence of growth rates on monomer concentration 
 also excludes fragmentation as a mechanism for generating additional 
 growth sites since the latter depends on aggregate but not monomer 
 concentration . the absence of fragmentation 
 is also evident from the lack of disintegration of isolated protofibrils 
 when dissolved in buffer ( figure 1 ) . finally , 
 the lack of a finite threshold to activate the self - replication process 
 is at odds with any nucleated process since the latter requires a 
 finite driving force ( i.e. , supersaturation ) . the inability to induce 
 amyloid formation by native monomers at any concentration in the absence 
 of seeds ( figure 3a ) , the lack of a lag phase 
 for oligomer formation under denaturing ( seed growth ) conditions , 
 and the immediate onset of growth from native monomers upon even modest 
 seed addition , all imply that variants of nucleated polymerization 
 and nucleated conformational conversion , by themselves , are difficult 
 to reconcile with the growth process reported here . these discrepancies 
 with existing models compel us to propose a new 
 model for oligomeric fibril growth from native monomers . the essential 
 features this model attempts to incorporate are a lack of spontaneous 
 oligomer formation from native monomers , exponential growth when seeding 
 native monomers with preformed oligomers , no discernible threshold 
 for the onset of oligomer formation , the presence of a nucleation 
 barrier for protofibril formation , and the previously reported morphological and structural similarities of oligomers and 
 protofibrils . these features are consistent 
 with two distinct  activities  of oligomers and protofibrils 
 that operate  orthogonal  from each other : self - replication 
 of oligomers from native monomers via a mechanism resembling a bimolecular 
 reaction combined with nucleated polymerization of protofibrils from 
 oligomers which resembles a nucleated phase separation . figure 8 summarizes the aggregation behavior for oligomers / protofibrils 
 vs those of filaments at denaturing vs native temperatures . at denaturing 
 temperatures , amyloid filament growth proceeds via nucleated polymerization , 
 with likely contributions from secondary generation . at physiological 
 temperatures , these filaments are stable against dissociation or fragmentation , 
 but can neither nucleate nor grow from native monomers . along the 
 oligomeric assembly pathway partially denatured monomers undergo barrier - free 
 self - assembly into oligomers which , in turn , undergo nucleated polymerization . it should be noted that protofibril nucleation does not leave a clear 
 signature in either tht or sls kinetics , but is readily apparent in 
 dls . at physiological temperatures , spontaneous 
 formation of oligomers from monomers at even the highest concentrations 
 is not observed , arguing further against a phase separation process . upon addition of preformed and stabile oligomers ( or protofibrils ) , 
 their self - replication from native monomers emerges . under denaturing 
 conditions the ability for self - replication similar 
 to enzymes acting on their substrate , this autocatalytic self - replication 
 mechanism has no discernible substrate threshold . self - replication , 
 in turn , accounts for the lack of a saturation threshold at high substrate 
 concentrations . we further surmise that the subsequent polymerization 
 of protofibrils uses oligomers as the growth substrate and proceeds 
 at a site distinct from that for self - replication . based on the identical 
 cross - sectional areas of oligomers and protofibrils measured with 
 calibrated afm we have previously argued that oligomers , not monomers , 
 are the growth substrate for lysozyme protofibrils . with oligomers as their growth substrate , protofibrils 
 can only continue to elongate in the presence of native monomers if 
 they provide two distinct sites , one for generating oligomers from 
 native monomers and another for using them as a substrate for elongation . this uncoupling of activities is also supported by the observation 
 that protofibril formation is a nucleated process while replication 
 of oligomer from either oligomeric or protofibril seeds will proceed 
 without a threshold . schematic of the free energy landscape 
 for oligomeric ( left ) vs filamentous ( right ) amyloid growth at denaturing 
 ( top ) vs physiological ( bottom ) temperatures . at denaturing 
 temperatures , oligomers and protofibrils utilize denatured monomers 
 as growth substrate . oligomers , in turn , become the substrate for 
 subsequent nucleation and polymerization of protofibrils . at physiological 
 temperatures preformed oligomers , but not filaments , can self - replicate from native 
 monomers . studies 
 of amyloid fibril assembly under controlled in 
 vitro conditions are critical for unraveling the molecular 
 mechanisms underlying amyloid fibril formation in vivo . our current understanding of molecular aggregation mechanisms , 
 however , is mostly derived from in vitro studies 
 under fixed solution conditions which , in the case of native proteins , 
 require partial denaturation . computations 
 with model peptides in two and three dimensions have suggested that 
 amyloid aggregation might proceed utilizing different conformations 
 of monomers and aggregates , which can interact in distinct ways upon 
 changes in solution environment ( temperature , ph , ionic strength , 
 concentration ) . this implies that mechanisms 
 for amyloid self - assembly observed under partially denaturing conditions 
 might not be fully representative of those active under physiological 
 conditions . our observations on lysozyme amyloid formation at denaturing 
 vs physiological temperatures do indeed reveal significant changes 
 to the thermodynamic stability , solubility , rates of fragmentation , 
 and aggregation propensity of oligomeric vs filamentous amyloid aggregates . they particularly suggest that oligomer formation at denaturing vs 
 physiological temperatures switches from the spontaneous self - assembly 
 of partially denatured monomers to autocatalytic self - replication 
 from native monomers . lowering solution temperatures into the 
 physiological range resulted 
 in either dramatic reduction of dissociation / fragmentation or self - association 
 rates , or in complete stabilization for each of the amyloidogenic 
 hew - l aggregates . yet , tau filaments generated at high temperatures readily dissociated 
 at physiological temperatures unless they were stabilized by an extrinsic 
 cofactor such as heparin . the emergence 
 of the thermodynamic stability of oligomers at physiological ph and 
 temperature is intriguing since oligomers are typically considered 
 short - lived transients or metastable intermediates of the amyloid 
 assembly process . similarly , the loss 
 of solubility of all aggregates upon increasing solution ph , while 
 not surprising , further highlights that the properties of various 
 templates also depend on the conditions they were generated under . our observations suggest that the solubility and mechanical stability 
 of all amyloid species are highly sensitive to their solution environment . the gain of stability of amyloid aggregates under physiological conditions 
 is a property that is clearly relevant for understanding how these 
 aggregates might spread to other cells and tissues and , potentially , 
 promote formation of new amyloid growth there . changes in solubility 
 of amyloid aggregates with solution environment is equally germane 
 for our understanding of functional amyloids . for example , peptide hormones stored 
 as amyloids in the low - ph environment of dense - core vesicles have 
 to remain stable , but need to dissolve rapidly upon release into the 
 bloodstream . most significantly , 
 though , is the ability for self - replication 
 that emerged at physiological temperatures . surprisingly , only oligomers 
 and protofibrils were able to induce autocatalytic self - replication 
 from natively folded lysozyme . the long rigid filaments considered 
 most  representative  of amyloid aggregates , in contrast , 
 remained inert at physiological temperatures . this is in stark contrast 
 to the behavior of filaments at partially denaturing temperatures . thermally denatured lysozyme monomers represent favorable substrates 
 for the nucleation and growth of amyloid aggregates along either assembly 
 pathway . rapid filaments growth at denaturing 
 temperatures vs their lack of growth at native temperatures implies 
 that filaments require denatured monomers as a growth 
 substrate . conversely , the ability of oligomers and protofibrils , 
 but not filaments , to seed growth at native temperatures highlights 
 that seeded amyloid formation by native proteins depends not only 
 on monomer stability and structure but on the stability and structure 
 of amyloid templates and their interactions with their monomeric substrate . it further implies that only a subset of amyloid aggregates might 
 have the capacity for autocatalytic self - replication required for 
 their prion - like propagation in tissue . it is worth 
 reiterating that there is no sign of spontaneous oligomer formation 
 at native solution conditions ( figure 3a ) . 
 under the denaturing conditions used for seed growth , barrier - free 
 assembly from denatured monomers hence , the capacity of lysozyme amyloid oligomers and protofibrils 
 for self - replication only becomes apparent at physiological conditions 
 where spontaneous self - assembly is abrogated . are there any 
 hints regarding how structural differences might 
 account for the observed capacity for self - replication for oligomers 
 and protofibrils but not filaments ? we have previously shown that 
 lysozyme filaments , oligomers , and protofibrils all develop -sheet 
 peaks in the  amyloid - band  of their ftir spectra . therefore , they all represent amyloid - like aggregates , albeit with 
 distinguishable structural fingerprints . specifically , the slight differences in the peak positions for filaments 
 vs oligomers near 1620 cm and the weak shoulder 
 for oligomers / protofibrils near 1695 cm both hint 
 at underlying structural differences between these two aggregate species . 
 these differences might well correlate with the -sheet - rich 
 but distinct structures recently reported for amyloid filaments vs 
 oligomers reported for model peptides . however , we presume that not all oligomers are created 
 equal . this is suggested by variations in the toxicity for oligomers 
 from the n - terminal of bacterial hypf when generated at different 
 ph s . in contrast to the -sheet 
 enriched oligomers in this study , liquid - like disordered oligomers 
 have been implicated to underlie nucleated conformational conversion 
 of poly - q . the ability of oligomers and 
 their polymers for self - replication from native protein further adds 
 to the growing list of pathogenic features ascribed to this class 
 of amyloid aggregates . it also reproduces 
 one defining characteristic of prion proteins : in vitro self - replication . the structural features that 
 imbue lysozyme oligomers with the capacity for self - replication are 
 not obvious , and whether this the data on seeded amyloid growth with native lysozyme also suggest 
 that the observed self - replication and polymerization of amyloid oligomers 
 requires a new molecular model for oligomeric amyloid growth . figure 8 summarizes how 
 we envision the temperature - induced changes in the aggregation behavior 
 along the filamentous and oligomeric assembly pathways seen in our 
 experiments . as outlined above , neither templated assembly nor nucleated 
 polymerization with inclusion of secondary mechanisms nor nucleated 
 conformational conversion is fully consistent with the kinetics of 
 oligomeric amyloid growth under native conditions . our proposed model 
 of  orthogonal autocatalytic self - replication and nucleated 
 polymerization  shares important similarities with basic aspects 
 of these existing models . most prominently , self - replication provides 
 a mechanism for spawning new aggregates similar to secondary nucleation 
 and , thereby , generates exponential growth . in addition , we expect 
 the nucleated polymerization of protofibrils to replicate most aspects 
 of the current models of nucleated polymerization of filaments with 
 a secondary nucleation pathway . first of all , oligomer 
 self - replication proceeds without a discernible lower threshold concentration 
 of monomers ( saturation concentration ) . this implies that self - replication 
 has the characteristics of an autocatalytic chemical reaction instead 
 of a phase separation phenomenon ( figure 7b ) . the lack of saturation at high monomer concentrations , in turn , is 
 a simple consequence of the autocatalytic feedback cycle in which 
 the product of one replication cycle serves as the  enzyme  
 in the next cycle . furthermore , the growth substrate for oligomer 
 formation and protofibril elongation in our model are two distinct 
 species : native monomers are the substrate for oligomer self - replication 
 while protofibril nucleation and polymerization utilize oligomers . the separation of self - replication and elongation allows these two 
 processes to proceed in a coordinated fashion that feeds back onto 
 each other . overall , our results suggest that the process of 
 amyloid fibril 
 assembly , while displaying various generic aspects , might proceed 
 through a variety of different pathways . multiple assembly mechanisms 
 become dominant not just by changes in monomer structure but by the 
 type of amyloid aggregate formed and its specific propensity to interact 
 with various monomeric conformations under various solution conditions . such a diversity of mechanisms might help explain the wide variety 
 of phenotypes and aggregation behavior reported for both pathological 
 and functional amyloids .
self - assembly 
 of amyloid fibrils is the molecular mechanism best 
 known for its connection with debilitating human disorders such as 
 alzheimer s disease but is also associated with various functional 
 cellular responses . 
 there is increasing evidence that amyloid formation 
 proceeds along two distinct assembly pathways involving either globular 
 oligomers and protofibrils or rigid monomeric filaments . 
 oligomers , 
 in particular , have been implicated as the dominant molecular species 
 responsible for pathogenesis . yet 
 the molecular mechanisms regulating 
 their self - assembly have remained elusive . 
 here we show that oligomers / protofibrils 
 and monomeric filaments , formed along distinct assembly pathways , 
 display critical differences in their ability to template amyloid 
 growth at physiological vs denaturing temperatures . at physiological 
 temperatures , 
 amyloid filaments remained stable but could not seed 
 growth of native monomers . 
 in contrast , oligomers and protofibrils 
 not only remained intact but were capable of self - replication using 
 native monomers as the substrate . 
 kinetic data further suggested that 
 this prion - like growth mode of oligomers / protofibrils involved two 
 distinct activities operating orthogonal from each other : autocatalytic 
 self - replication of oligomers from native monomers and nucleated polymerization 
 of oligomers into protofibrils . 
 the environmental changes to stability 
 and templating competence of these different amyloid species in different 
 environments are likely to be important for understanding the molecular 
 mechanisms underlying both pathogenic and functional amyloid self - assembly .