Myostatin Amyloid Formation Figure 3. Characterisation of MstnPP soluble aggregates by negative-stain transmission electron microscopy (TEM) and ThT binding. (A–B) TEM of MstnPP soluble aggregates and C. insulin positive control containing both protofibrils (black arrow) and mature fibrils (white arrow). D. ThT binding of MstnPP soluble aggregates (green), compared to an insulin positive control (red), the MstnPP dimer (yellow) and a buffer (50 mM Tris-HCl pH 8.5, 150 mM NaCl) blank (blue). doi:10.1371/journal.pone.0009170.g003 stacking gel owing to its extremely large size. Following incubation with trypsin this property is maintained even after overnight incubation (18 h) at 37uC and a 5-fold increase in trypsin concentration (MstnPP:trypsin 20:1) (Fig. 6C). Inability to enter the stacking gel is not a definitive indication of proteolysis resistance since partial hydrolysis may result in products smaller than fibrils and/or protofibrils yet still large enough to be retained in the stacking gel. To address this possibility, TEM analysis revealed that both MstnPP protofibrils (Fig. 6Di) and mature fibrils (Fig. 6Dii) had unchanged morphology after trypsin incubation. MstnPP Aggregates Exhibit Amyloid Characteristics after Incubation at 37uC The formation of amyloid fibrils by MstnPP at 60uC and pH 5.3 is comparable to conditions routinely used to promote rapid amyloid formation in in vitro model systems for amyloid diseases. To investigate the temperature range at which structural changes occur, MstnPP aggregates were analysed by CD from 10 to 65uC immediately after suspension in pH 5.3 (Fig. S3). The spectrum had changed significantly by 45uC with a loss of a-helix beginning from 30uC and the appearance of a characteristic amyloid b-sheet-rich spectrum at 65uC. Subsequently, MstnPP aggregates were incubated at 37uC at pH 5.3 and monitored with ThT binding, CD spectroscopy and TEM (Fig. 7). After overnight incubation ThT binding increased only slightly (Fig. 7A) and CD indicated that b-sheet aggregation has occurred (Fig. 7B). Under TEM, the 37uC protofibrils have a similar morphology to the 60uC samples, however aggregation is less extensive (Fig. 7C); elongation, clustering and ring-like structures are apparent. After one week of incubation at 37uC, ThT binding increased significantly (Fig. 7A) and CD showed a definite b-sheet transition (Fig. 7B). TEM images show increased aggregation, lateral association and a granular to smooth morphology transition (Fig. 7D). Although fibril formation did not occur at 37uC in the time period observed for incubation at 60uC, it is likely that the kinetics of fibril formation will be slower at the lower temperature. However, these results show that the properties of protofibrils, proposed to be direct precursors of mature amyloid fibrils, have been adopted at 37uC; amyloid formation is likely to occur over an extended period of time. MstnPP Aggregates Have a Negative Effect on C2C12 Myoblast Viability Oligomeric species in the amyloid formation pathway of a number of proteins are cytotoxic when added to the media of cultured cells [41,42,65]. The effect of MstnPP soluble aggregates, PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9170